Particle settling devices

ABSTRACT

The present disclosure relates to settling devices for separating particles from a bulk fluid with applications in numerous fields. The particle settling devices of the present disclosure may include a stack of truncoconical cones that may be arranged in opposite orientation, apex to base. Other embodiments include several concentric vertical tubes attached to conical surfaces at the bottom, with inclined settling strips attached to the vertical tubes in annular regions between the tubes. These devices are useful for separating small (millimeter or micron sized) particles from a bulk fluid with applications in numerous fields, such as biological (microbial, mammalian, plant, insect or algal) cell cultures, solid catalyst particle separation from a liquid or gas and waste water treatment.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/586,902, filed May 4, 2017, now U.S. Pat. No. 10,596,492 with anissue date of Mar. 24, 2020, which is a continuation-in-part of U.S.patent application Ser. No. 15/324,062, filed Jan. 5, 2017, and U.S.Pat. No. 10,596,492 is a continuation-in-part of PCT Application No.PCT/US2015/063195 having an international filing date of Dec. 1, 2015and which designated the United States, and U.S. Pat. No. 10,596,492also claims priority under 35 U.S.C. § 119(e) to U.S. Provisional PatentApplication No. 62/332,546, filed May 6, 2016, and to U.S. ProvisionalPatent Application No. 62/459,509, filed Feb. 15, 2017. U.S. patentapplication Ser. No. 15/324,062 is a national stage application under 35U.S.C. 371 of PCT Application No. PCT/US2015/039723 having aninternational filing date of Jul. 9, 2015, which designated the UnitedStates, which PCT application claims the benefit of U.S. ProvisionalPatent Application No. 62/022,276, filed Jul. 9, 2014, and to U.S.Provisional Patent Application No. 62/037,513, filed Aug. 14, 2014. PCTApplication No. PCT/US2015/063195 claims the benefit of U.S. ProvisionalPatent Application No. 62/086,122, filed Dec. 1, 2014. All of theseapplications are incorporated herein by reference in their entirety.

FIELD

This disclosure provides cell or particle settling devices with enhancedsettling on multilayered inclined surfaces. The devices of the presentdisclosure have applications in numerous fields, including: (i) highcell density biological (mammalian, microbial, plant or algal) cellcultures secreting polypeptides, hormones, proteins or glycoproteins,vaccines or vaccine-like particles, or other small chemical products,such as ethanol, isobutanol, isoprenoids, flavor and fragrancecompounds, etc.; (ii) separating and recycling porous or non-poroussolid catalyst particles catalyzing chemical reactions in liquid or gasphase surrounding solid particles; (iii) separating and collecting newlyformed solids in physical transformations such as crystallization,flocculation, agglomeration, precipitation, etc., from the surroundliquid phase; (iv) capture and purification of secreted proteins, suchas monoclonal antibodies, and others, on affinity ligands, such asprotein A immobilized on microspherical beads; (v) in vitro expansion ofvarious mammalian cells, such as human mesenchymal stem cells,differentiated human cells e.g. cardiomyocytes or red blood cells,modified human cells, e.g. chimeric antigen receptor transfected Tlymphocytes or CAR-T cells, etc. for autologous or allogenic celltherapy applications; and (vi) clarifying process water in large scalemunicipal or commercial waste water treatment plants by settling andremoving complex biological consortia or activated sludge or other solidparticles.

DESCRIPTION OF RELATED ART

Of all the above-mentioned fields of application for settling devices,the more immediately applicable well-established field is the productionof biological proteins, polypeptides or hormones secreted fromsuspension cultures of recombinant microbial or mammalian cells. Mostcommon methods of producing biological proteins in recombinant mammalianand microbial cells rely on fed-batch cultures, wherein cells are grownto high cell densities and then typically exposed to an induction mediumor inducer to trigger the production of proteins. If the desiredproteins are secreted out of the cells, it is more profitable to switchfrom a fed-batch culture to a continuous perfusion culture, which canmaintain high cell density and high productivity over a much longerduration of culture. During continuous perfusion cultures, live andproductive cells are retained or recycled back to the bioreactor whilethe secreted proteins are continuously harvested from the bioreactor fordownstream purification processes.

Some key advantages of continuous perfusion cultures over fed-batchcultures are: (1) the secreted protein products are continuously removedfrom the bioreactor, without subjecting these products to potentialdegradation by proteolytic and/or glycolytic enzymes released into theculture medium from dead cells; (2) live and productive cells areretained or recycled back to achieve high cell densities in continuousperfusion bioreactors, where they continue to produce valuable proteinsinside the controlled bioreactor environment for much longer cultureduration, rather than being killed and removed from the bioreactor atthe end of each fed-batch culture; (3) the perfusion bioreactorenvironment can be maintained much closer to steady state conditions(thereby maintaining a more consistent product quality by design) withthe continuous addition of fresh nutrient media and removal of wasteproducts along with the harvested protein products, unlike thedynamically changing concentrations of nutrients and waste products infed-batch culture; and (4) with a subset of cell retention devices,smaller dead or dying cells can be selectively removed from theperfusion bioreactor before these cells lyse and release theirintracellular enzymes, thereby maintaining a high viability fraction ofcells and high quality of the secreted protein products as they areharvested.

Many cell retention devices have been developed in the mammalian cellculture industry, such as the internal spin filter devices (Himmelfarbet al., Science 164: 555-557, 1969), external filtration modules(Brennan et al., Biotechnol. Techniques, 1 (3): 169-174, 1987), hollowfiber modules (Knazek et al., Science, 178: 65-67, 1972), gravitationalsettling in a cyclone (Kitano et al., Appli. Microbiol. Biotechnol. 24,282-286, 1986), inclined settlers (Batt et al., Biotechnology Progress,6:458-464, 1990), continuous centrifugation (Johnson et al.,Biotechnology Progress, 12, 855-864, 1999), and acoustic filtering(Gorenflo et al., Biotechnology Progress, 19, 30-36, 2003). The cycloneswere found to be incapable of producing enough centrifugal force forsufficient cell separation at the device sizes and harvest flow ratesused in the mammalian cell culture experiments (Kitano et al., 1986) andmammalian cells are seriously damaged at higher flow rates (andcentrifugal forces) necessary for efficient cell separation (Elsayed, etal., Eng. Life Sci., 6: 347-354, 2006). While most of the other devicesadequately retain all mammalian cells from the harvest, these devicesare unable to separate dead cells from the live cells desired in thebioreactor. Consequently, dead cells keep accumulating inside theperfusion bioreactor and the membrane filters get clogged, necessitatingthe termination of the continuous perfusion bioreactor, typically withinthree or four weeks of mammalian cell culture.

Among all the cell retention devices available today, only the inclinedsettlers (Batt et al., 1990, supra and Searles et al., BiotechnologyProgress, 10: 198-206, 1994) enable selective removal of smaller deadcells and cell debris in the overflow or harvest stream, while bigger,live and productive mammalian cells are continually recycled via theunderflow back to the perfusion bioreactor. Therefore, it is feasible tocontinue the perfusion bioreactor operation indefinitely at highviability and high cell densities while the protein product iscontinuously harvested from the top of the inclined settler.

The inclined settler has previously been scaled up as multi-plate orlamellar settlers (Probstein, R. F., U.S. Pat. No. 4,151,084, April1979) and used extensively in several large-scale industrial processessuch as wastewater treatment, potable water clarification, metalfinishing, mining and catalyst recycling (e.g. Odueyngbo et al., U.S.Pat. No. 7,078,439, July 2006).

Citing our first demonstration of a single plate inclined settler (Battet al., 1990) to enhance productivity of secreted proteins in mammaliancell culture applications, a multi-plate or lamellar settler device hasbeen patented for the scale up of inclined settlers for use in hybridomacell culture (Thompson and Wilson, U.S. Pat. No. 5,817,505, October1998). Such lamellar inclined settler devices have been used to culturerecombinant mammalian cells in continuous perfusion bioreactors at highbioreactor productivity (due to high cell density) and high viability(>90%) for long durations (e.g. several months without any need toterminate the perfusion culture). U.S. Patent Publication No.2011/0097800 to Kauling et al., describes a scaled up version ofinclined settlers that uses cylindrical tubes wrapped at inclinedangles. The device is described as useful in the culturing of largermammalian cells, such as CHO, BHK, HEK, HKB, hybridoma cells, ciliates,and insect cells.

None of these cell retention devices have been demonstrated forharvesting secreted protein products in perfusion bioreactor cultures ofthe smaller, and hence more challenging, microbial cells. Lamellarsettlers have been tested with yeast cells to investigate cell settlingwith limited success (Bungay and Millspaugh, Biotechnology andBioengineering, 23:640-641, 1984). Hydrocyclones have been tested inyeast suspensions, mainly to separate the yeast cells from beer, againwith only limited success (Yuan et al., Bioseparation, 6: 159-163, 1996;Cilliers and Harrison, Chemical Engineering Journal, 65: 21-26, 1997).

A modified cyclone with a spiral vertical plate inside the cyclone wasproposed to improve the separation efficiency in wastewater treatment(Boldyrev V V, Davydov E L settling tanks, as described in RussianPatent No. 2,182,508) and an earlier description of this arrangement hasbeen described for the decantation of solids in liquid suspension (U.S.Pat. No. 4,048,069, September 1977). The modified cyclone disclosed inRussian patent No. 2,182,508 includes a spiral wound plate housed in avertical cylindrical barrel with a conical bottom. A slit is providedalong the entire height of a central waste water inlet tube, which isplugged at the bottom in order to channel waste water from the inlettube into the vertical spiral wound plate. The spiral starts at thecentral tube and ends at the wall of the cylindrical housing, forming achannel through which particle-laden waste water flows. The particlessettle in the vertical sedimentation column of the spiral channel. Theheight of the settler zone is the vertical height of the spiral plateand the width of the channel is formed by the walls of the spiral woundplate, which is held constant throughout its length. A pipe for removingthe purified water is installed at the upper part of the cylindricalbody. A conduit for removing sediment is installed at the bottom of theconical bottom portion. In operation, waste water enters through thecentral tube and enters the spiral zone through the slit or opening. Thespiral channel serves to increase the flow path and hence increase theresidence time of liquid in the settler. The spiral also serves toincrease the contact (wall) area for the fluid. The main driving forcein clarification is gravity acting on the particles of the suspension,as the suspension goes around the spiral-wound vertical sedimentationcolumn. The slurry that is left on the wall of the spiral or in thechannel, falls into the conical bottom of the settler, and is removedperiodically from the settler. Purified water is drawn from a pipe onthe side of cylindrical housing near the top.

As described in the Russian patent document, the flow pattern of thewaste water-containing solids is reversed from the typical flow patternof a common cyclone, as the dirty water enters at the center, via thecentral tube and enters into the spiral channel through the slits, andthe purified water is removed from the periphery or outside of thevertical cylindrical body via a purified water pipe. This modified andflow-reversed cyclone device has not been proposed for, or applied toany fields other than waste water treatment.

Thus, a particle settling device that can leverage centrifugal forcesand gravitational forces on particles in liquid suspension in arelatively small space is desired.

SUMMARY

This disclosure provides cell or particle settling devices with enhancedsettling on multilayered, inclined surfaces arranged within a cyclonehousing. The particle separation devices of this disclosure may be usedin numerous applications, and represent a large improvement over theprior art separation devices. In some embodiments these surfaces may beattached to a plurality of vertical cylindrical plates. In otherembodiments, the settling devices include a spiral conical surface, orseveral inclined plates approximating an angled conical surfaceconnected to the bottom of a spiral. The numerous, layered inclinedplates enhance the settling efficiency of the particles from the bulkfluid moving either downward or upward inside a conical cyclone assemblyin which the liquid volume moves progressively from the periphery of theconical spiral to the center of the settler device.

In one or more embodiments, the settler devices of this disclosureinclude a cyclone housing (often referred to as a “hydrocyclone”), aspiral vertical plate positioned inside the cyclone housing, the spiralvertical plate joined at its bottom with a spiral conical surfacetapering down to an opening. Notably, in some embodiments, there is noplug or other impediment preventing the flow of liquid or suspendedparticles from the spiral vertical plates or spiral conical surfacestoward the opening. The spiral conical surface forms lamellar inclinedsettler plates in a conical geometry.

In related embodiments, the devices of the disclosure include a cyclonehousing, a spiral vertical plate positioned inside the cyclone housing,the spiral vertical plate joined at its bottom with a spiral conicalsettling surface tapering down to an opening. In this embodiment, thevertical spiral plate has a decreasing height towards the center of thedevice, and substantially constant spacing between the successive spiralrings. The spiral conical settling surfaces at the bottom of a spiralvertical plate have increasing lengths to match the decreasing height ofthe vertical spiral plate and extend to approximately the center of thesettler device. Similarly, in some embodiments, there is no plug orother impediment preventing the flow of liquid or suspended particlesfrom the spiral vertical plates or spiral conical surfaces toward theopening.

In one embodiment, the inclined settling surfaces are provided bynumerous annular strips, or ‘ramps’, of metal stretched and aligned atan angle between about 30 degrees and about 60 degrees from vertical,and joined to the outer surface of each cylinder or tube. In anotherembodiment, the angle of the ramps is about 45 degrees from thevertical. The horizontal spacing between the successive parallel rampsin each annular region between the cylinders can be varied between about5 mm to about 15 mm.

The inclined settling strips significantly enhance the settlingefficiency of the particles from the bulk fluid as the bulk fluid movesupward in the annular settling zones created between the vertical tubes.As the harvest moves up through the annular inclined settling zones,bigger particles (e.g., live and productive cells) settle on the strips,slide down, exit at the outer edges of the strips and fall downvertically into the conical section of the cyclone assembly. Thesedevices can be scaled up or down to suit the separation needs ofdifferent industries or applications or sizes as the separation surfaceis scaled up or down volumetrically in three dimensions, compared to themore typical one- or two-dimensional scaling of previous settlingdevices.

In all of the embodiments described above, attaching the spiral verticalplates to the spiral conical settling surfaces can be accomplished bywelding or otherwise joining (i.e., gluing or other adhesives, bonding,ultrasonic welding, clamping, or the like) curved angular plates at afixed inclination to the circular bottom edge of the spiral verticalplate.

In all of the embodiments described above, the spiral conical surfacecan be tightly fitted to obtain a continuous conical spiral surface.Alternatively, small gaps between the spiral conical surfaces areacceptable for a discontinuous conical spiral surface, provided the gapsin the surface are staggered between successive conical spirals.

In all of the embodiments described above, the angle of inclination forthe conical spiral surfaces can be between 30 degrees and 60 degreesfrom the vertical. In certain embodiments, the angle of inclination forthe conical spiral surfaces is about 45 degrees from the vertical. Forstickier particles (typically mammalian cells), the angle of inclinationis preferably closer to the vertical (i.e., about 30 degrees from thevertical. For non-sticky solid particles (for example, catalystparticles), the angle of inclination can be further from the vertical(preferably, about 60 degrees from vertical).

In other embodiments, the settler device of this disclosure includes acyclone housing that encloses a series of stacked cones positionedinside the cyclone housing, tapering down to a central opening, with novertical plates. The cones of this embodiment are supported in thestack, one above the other, by supports that maintain a distance (orchannel width) between the successive cones in the stack. In certainembodiments, the supports comprise three or more projections attached tothe upper and/or lower surface of one or more of the cones to positionsuccessive cones at a desired distance (the desired channel width)apart. Optionally, the supports may comprise at least three L-shapedelements interconnected to a surface of each cone that is distal to thetruncated apex of the cone. The L-shaped elements include a first sideinterconnected to a second side at an apex and are interconnected to thesurface such that the first side supports a second cone in the stack ofcones. The second side is substantially parallel to the surface of thecone. Optionally, the second side may project beyond the cone to spacethe cone from an interior surface of the cyclone housing. As in theprevious embodiments, in some embodiments, there is no plug or otherimpediment preventing the flow of liquid or suspended particles from thestacked conical surfaces toward the central opening.

In another embodiment, a settler devices of this disclosure includes acyclone housing enclosing:

1) a first stack of two or more stacked cones, each having a centralopening, and,

2) an optional second stack of two or more stacked cones, each having acentral opening, joined at or near their bottom with conical surfacestapering down to a central opening at the bottom of the cyclone housing.

The stacked cones (in both the first and optional second stack of two ormore stacked cones) comprise at least three projections supporting eachcone above the next successive cone in the stack. The projections arepreferably placed at a substantially constant distance and are formed ata generally equal size to hold each successive cone in the stack atabout an equal spacing between all of the cones in the stacks. In oneembodiment, there are at least three projections for each cone toproperly support each successive cone, but each cone may comprise morethan three projections, as needed to adequately or properly support thecone. For example, each cone may comprise four projections, or maycomprise eight projections, to support the next successive cone in thestack.

The projections, or “vertical supports,” may represent an impediment tosettled particles or cells sliding down the surface of a cone towardsthe central opening or the gap around the inner circumference of thecyclone housing between the housing and the cones. These projections areattached to one surface of a cone, but these projections do not attachto another cone in a stack of cones. Thus, these projections do notattach two or more cones in a stack to one another.

There is preferably a substantially constant spacing between eachsuccessive conical surface created by the projections supporting eachsuccessive cone in a stack of cones. The spacing between successivecones may be varied between about 1 mm to about 2.5 cm.

This arrangement of settling surfaces, provided by the successive stacksof cones, each of which is supported by the next successive cone, but isnot permanently attached to the next successive cone, is particularlyuseful for separation applications in which the particle settlingdevice, and the conical surfaces therein, requires regular or continualservice, such as disassembly and cleaning of the conical settlingsurfaces within the settler device.

This arrangement of first and optional second stacks of conessignificantly enhances the settling efficiency of particles from a bulkfluid as the bulk fluid moves through the settling device. As the bulkliquid, including particles such as cells, moves through the stackedcones of the settler device of this disclosure, bigger particles (e.g.,live and productive cells) settle on the surface of the cones. Cellssliding down the upper or first stack of cones, slide down the conicalsurfaces to the outer edges of the cones and fall down vertically intothe conical section of the cyclone housing. Additionally, cells slidingdown the lower or second stack of cones, slide down the conical surfacesto the central opening of the cones and fall down vertically towards thecentral opening of the cyclone housing.

These devices can be scaled up or down to suit the separation needs ofdifferent industries or applications or sizes as the separation surfaceis scaled up or down volumetrically in three dimensions, compared to themore typical one- or two-dimensional scaling of previous settlingdevices.

Scale up of the devices of this disclosure can be performed simply byincreasing the diameter of the cyclone housing (and correspondinglyincreasing the diameter of cones stacked inside) and/or increasing theheight of the cyclone (which increases the number of cones in either oneor both of the first and second stack of cones). For example, using a10-inch (25.4 cm) diameter cylinder, with a spacing of approximately 10mm between successive ramps, about 80 ramps going up may be welded tothe outside of the 10-inch (25.4 cm) diameter cylinder. For a 12-inch(30.5 cm) diameter cylinder, approximately 92 inclined settling rampscan be placed within the cylinder, and so on, in proportion. Theeffective projected area for cell settling increases proportional to thesquare of the diameter of cyclone housing and increases proportional tothe height of internal cylinders. So the effective settling area of thecompact settling devices of this disclosure scales up proportional tothe cube of cyclone diameter (assuming the height of the internalsettler is also increased proportionally) or equivalently, to the volumeof cyclone housing. This three dimensional or volumetric scale-up of theeffective settling area makes the settling device of this disclosuremuch more compact compared to previous inclined settler devices.

The radial spacing in the annular regions between different cylinderscan be between about 1 cm to about 10 cm, with an optimum around about2.5 cm. A small clearance of about 1 mm between the inclined settlingramps and the internal surface of the next successive cylinder providesuseful space for settled particles (for example cells) to slide down thesurface of the ramps and exit the ramps on the side, rather than slidingall the way down to the bottom of the ramp. The side-exiting cellssettle vertically along the inside of each cylinder. When these settlingcells reach the conical surface at the bottom of each cylinder, theyslide down on the inclined surface on the cone to the central opening atthe bottom of the cyclone housing. An advantage of the increasing fluidvelocity while going down the inclined conical surface to the centralopening is that the increasing number of settled cells sliding down thecone are swept down to the central opening, rather than being allowed toaccumulate by the faster liquid velocities.

In all of the embodiments of the settler devices of this disclosure, thecomponents of the settler devices may be composed of a metal and/or aplastic. In certain embodiments, the components of the settler devicesare composed of stainless steel (such as stainless steel alloy 316 orsimilar materials used for applications in microbial or mammalian cellculture, as well as other metals used for applications in chemicalprocess industries, such as catalyst separation and recycle). Inspecific embodiments these settler devices are composed entirely ofstainless steel. In specific embodiments including a spiral verticalplate, and the spiral conical surface and the spiral vertical plate aremetals joined by welding.

Metal settling devices of this disclosure can be constructed by formingthe cyclone housing and forming the number of desired cones constitutingeach of the first and optional second stack of cones, sized to fitwithin the cyclone housing. Projections of the desired size, shape, andnumber may then be mounted on one or both surfaces of each cone in orderto support the next successive cone. In some embodiments, metal settlingdevices can be constructed by cutting out annular strips from a flatmetal sheet, and stretching them in a perpendicular direction to reachan angle between about 30 degrees and about 60 degrees (preferably about45 degrees) from vertical around an inner cylinder, and welding tabs atthe ends of the metal annular strips to the outside of the cylinder.

In other embodiments, the material of construction of these settlerdevices may also include non-metals including, for example, plastics foruse as single-use disposable settler devices, or glass for housingtransparency, etc. Optionally, in one embodiment, the settler devicesare composed entirely of plastic. A plastic settling device according tothis disclosure can be fabricated continuously, as a single piece,using, for example, injection molding or three-dimensional printingtechnologies. In some embodiments, the plastic settling device may betransparent or translucent. In one embodiment, at least a portion of thesettling device is translucent or transparent. In still anotherembodiment, the material of at least a portion of the settling device istransparent to light of a predetermine range of wavelengths.

The angle of inclination for the conical surfaces (or “ramps”) may bebetween about 30 degrees to about 60 degrees from the vertical. For usewith stickier particles (typically mammalian cells), the angle ofinclination may be closer to the vertical (i.e., around 30 degrees fromvertical). For use with non-sticky solid catalyst particles, the angleof inclination can be further from vertical (for example, around 60degrees from vertical).

In all embodiments, all or some of the surface of the cyclone housing,the spiral vertical plate or the conical surfaces may be completely orpartially coated with one or more of a non-sticky plastic, teflon, andsilicone. Additionally, or alternatively, the metals (especiallystainless steel) may be electropolished to provide a smooth surface.

One or more sensors may be positioned to monitor conditions within allembodiments of the settler devices of this disclosure. In oneembodiment, at least one sensor is positioned to monitor conditions witha tube or line interconnected to the settler devices of this disclosure.In another embodiment, the line is a return line interconnected to abottom outlet port of the settler device.

The sensors may be selected to determine one or more of pH, DO, Glucose,temperature, and CO₂ (include dissolved CO₂ which is also known aspartial CO₂) within the cyclone housing or the line. In one embodiment,the sensors include a probe in contact with a solution within thecyclone housing or the line. The probe may be affixed to an interiorsurface portion of the settler device or the line. In one preferredembodiment, at least one sensor and/or probe is positioned within thelower conical portion of the settler device. In another embodiment, thesensor and/or probe is spaced from one or more of the side port and thebottom port. In still another embodiment, the sensor and/or probe ispositioned within the line.

The probe may transmit data without contact to a reader. In this manner,the probe may measure a condition within the settler device and/or theline and transmit data to the reader outside the settler device. In oneembodiment, the probe is a fluorescent probe. One or more of pH, DO,Glucose, temperature, and CO₂ may be measured by the probe within thecyclone housing. The probe is affixed to a portion of the cyclonehousing. The portion of the cyclone housing is operable to transmitlight produced by the fluorescent probe. In one embodiment, the portionof the cyclone housing is transparent or translucent. The reader (ormeter) receives light from the fluorescent probe. In one embodiment, thereader includes an optical fiber that collects light transmitted by thefluorescent probe. Suitable probes and readers are available from avariety of vendors, including PreSens Precision Sensing GmbH.

In another embodiment, the probe within the settler device can transmitdata to the reader outside the settler device by a network connection.For example, in one embodiment the probe can communicate with the readerby WiFi, Bluetooth, or any other wireless communication modality.

All of the embodiments of the settler devices of this disclosure mayinclude a closure or lid over at least a portion of the cyclone housingat an end of the cyclone housing opposite the first opening. In all ofthese embodiments, the closure or lid may also include an outlet or portfor removing liquids or entering liquids into the settler device. In allof these embodiments, the opening and the additional ports or outlets inthe cylindrical housing and/or the lid are in liquid communication withthe outside and the inside of the cyclone housing to allow the passageof liquids into and/or out of the cyclone housing of the settler device,and in each instance of such opening or inlet/outlet, these passage waysinto and out of the cyclone housing may include valves or othermechanisms that can be opened or closed to stop or restrict the flow ofliquids into or out of the settler devices of this disclosure.

The thickness of the material constructing the cones is preferably asthin as necessary to maintain the rigidity of shape and to minimize theweight of the concentric stack of cones to be supported inside thecyclone housing. The radius and height of this device can be scaled upindependently as much as needed for the large-scale processes as may becalculated from predictive equations such as provided for inclined platesettlers (Batt et al. 1990, supra).

An important factor causing particle separation in the settler devicesof this disclosure is the enhanced sedimentation on the inclinedsurfaces, which has been successfully demonstrated by Boycott (Nature,104: 532, 1920) with blood cells and on inclined rectangular surfaces assuccessfully demonstrated by Batt et al. (1990) with hybridoma cellsproducing monoclonal antibodies. Additional factors enhancing thecell/particle separation are the centrifugal force on thecells/particles during their travel up the annular regions betweensuccessive cylinders and the settling due to gravity in the verticalsedimentation columns of the spiral channel.

While lamellar plates have been used to scale up inclined plate settlersby each dimension independently, i.e. increasing the length, or thewidth or the number of plates stacked on top of the each plate, thespiral conical settling zone can be scaled up in three dimensionssimultaneously by simply increasing the horizontal radius of thisdevice. As the horizontal radius of the device increases, the number ofvertical and conical surfaces can be proportionally increased by keepinga constant distance (or channel width) between the successive spirals.The particle separation efficiency is directly proportional to the totalprojected horizontal area of the inclined settling surfaces. With anincrease in device radius, the projected horizontal area increasesproportional to the square of the radius, and the number of feasiblespiral cones at a channel width also increases with the radius,resulting in a three dimensional scale up in the total projected area(i.e. proportional to the cube of radius) by simply increasing theradius.

Thus, a particle settling device of this disclosure may include acyclone housing and at least one vertical tube disposed inside thecyclone housing, the at least one vertical tube joined at one end with aconical surface tapering down to a first opening in the cyclone housing.At least one annular strip is attached to a vertical surface of the atleast one vertical tube at an angle between about 30 degrees and about60 degrees from vertical. There is at least one additional opening inthe cyclone housing substantially opposite the first opening. Thevertical tubes may include at least one material selected from the groupconsisting of a metal and a plastic. The vertical tubes may be stainlesssteel, and may be composed entirely of stainless steel. The verticaltubes, the annular strip, and the conical surfaces may all be metalsjoined by welding. Alternatively, the tubes may be composed entirely ofplastic. At least one surface of the cyclone housing, the at least onevertical tube, the annular strip, and the spiral conical surface iscoated with a non-sticky plastic or silicone.

The angle of inclination for the conical surfaces is about 45 degreesfrom vertical, or between about 30 degrees from vertical and about 60degrees from vertical.

The width of an annular ringed channel formed between adjacent verticaltubes is between about 1 mm and about 50 mm. The number of verticaltubes within the settler device may be between about 2 and about 30.

The settler device may include a closure over at least a portion of thecyclone housing at an end of the cyclone housing opposite the firstopening. At least one additional opening in the cyclone housing, may beconfigured to open from a side of the cyclone housing tangential to atleast one vertical tube, in liquid communication with the outside andthe inside of the cyclone housing.

A liquid harvest outlet may be formed in the closure, in liquidcommunication with the outside and the inside of the cyclone housing.

The annular strip is attached to the at least one vertical tube in aspiral that rises at an angle of about 45 degrees to vertical from anend of the tube adjacent the first opening spiraling around the at leastone vertical tube up to the opposite end of the at least one verticaltube. The annular strip may be attached to the at least one verticaltube and may be of sufficient width to leave a gap of between about 0.5mm and about 10 mm between an edge of the annular strip and the cyclonehousing or an adjacent vertical tube.

Thus, another aspect of this disclosure provides a method of settlingparticles in a liquid suspension. The method includes, but is notlimited to: (i) introducing a liquid suspension of particles into aparticle settling device of this disclosure; (ii) collecting particlesfrom a first opening in a cyclone housing of the settler device; and(iii) collecting a liquid from another opening in the settling device.In certain embodiments of this method, the liquid is collected from anopening in a closure that covers at least a portion of the cyclonehousing at an end of the cyclone housing opposite the first opening. Incertain embodiments, liquid is collected from at least one additionalopening in the cyclone housing, which opening is configured to open froma side of the cyclone housing.

In certain embodiments of these methods, the liquid suspension mayinclude at least one of a recombinant cell suspension, an alcoholicfermentation, a precipitating protein solution, a mixture of aqueousfluid containing cells and organic layer containing extracted organicproducts produced by the cells, a suspension of solid catalyst particlesin a liquid mixture containing mostly the products and depletedreactants, a suspension of microspheres coated with protein A moleculeswhich can bind the monoclonal antibodies from the cell culture broth, asuspension of microcarrier beads with mammalian cells growing attachedon the beads, a municipal waste water, and an industrial waste water. Incertain embodiments of these methods, the liquid suspension may includeat least one of mammalian cells, bacterial cells, yeast cells, plantcells, algal cells, human stem cells or differentiated human cellsand/or insect cells. In certain embodiments of these methods, the liquidsuspension may include at least one of biodiesel-producing algae cells,recombinant mammalian and/or murine hybridoma cells, metabolicallyengineered yeast cells producing secreted organic products, and yeast inbeer. In certain embodiments of these methods, the liquid suspension mayinclude recombinant microbial cells selected from Pichia pastoris,Saccharomyces cerevisiae, Kluyveromyces lactis, Aspergillus niger,Escherichia coli, and Bacillus subtilis.

In certain embodiments of these methods, the step of introducing aliquid suspension into the settler device includes directing a liquidsuspension from a plastic bioreactor bag into the particle settlingdevice.

In certain embodiments of these methods, the liquid collected from thesettler device may include at least one of biological molecules, organicor inorganic compounds, chemical reactants, and chemical reactionproducts. In certain embodiments of these methods, the liquid collectedfrom the settler device includes at least one of hydrocarbons,polypeptides, proteins, alcohols, fatty acids, hormones, carbohydrates,antibodies, antibodies, terpenes, isoprenoids, biodiesel, polyprenoids,and beer. In certain embodiments of these methods, the liquid collectedfrom the settler device includes at least one of biodiesel components,secreted therapeutic proteins or hormones such as insulin or itsanalogs, antibodies, monoclonal antibodies, brazzein, growth factors,sub-unit vaccines, viruses, virus-like particles, colony stimulatingfactors, erythropoietin (EPO), secreted flavor or fragrance compounds,including geraniol, myrcene, sweetener protein brazzein, etc.

Another aspect of the present disclosure is a particle settling devicethat may include, but is not limited to, a cyclone housing including oneor more of: (1) a first conical portion; (2) a second conical portion;(3) a cylindrical portion located between the first and second conicalportions; (4) at least one inlet for introducing a liquid into thecyclone housing; (5) a first outlet port; (6) a second outlet port; and(7) a first stack of cones located within the cyclone housing. In oneembodiment, the first outlet port is associated with the first conicalportion and the second outlet port is associated with the second conicalportion. Optionally, the liquid introduced into the cyclone housing maybe a liquid suspension including particles. The particles may be of aplurality of sizes.

In one embodiment, the first outlet port may be for harvesting aclarified liquid. The clarified liquid may include a first subset ofparticles. The first subset of particles may comprise cell debris, deadcells, and the like. Optionally, the first outlet port may be formed ina closure of the cyclone housing. The first outlet port being in liquidcommunication with the outside and the inside of the cyclone housing.

Optionally, in another embodiment, the second outlet port may be forharvesting a concentrated liquid. The concentrated liquid may include asecond subset of particles. In one embodiment, the second subset ofparticles may include live cells. In another embodiment, particles ofthe second subset of particles are generally larger than particles ofthe first subset of particles. In still another embodiment, eachparticle of the second subset of particles generally has a greater massthan the particles of the first subset of particles. The second outletport being in liquid communication with the outside and the inside ofthe cyclone housing.

In one embodiment, the first stack of cones occupies at least a portionof the first conical portion. Optionally, the first stack of conesoccupies at least a portion of the cylindrical portion. Optionally, oneor more cones of the first stack of cones includes a truncated apexoriented towards the first outlet port. Additionally, or alternatively,at least one cone of the first stack of cones is devoid of the centralopening. In another embodiment, each cone of the first stack of conesincludes an open base oriented towards the second outlet port. In oneembodiment, the cones of the first stack of cones are generally centeredin the cyclone housing. In another embodiment, the cones of the firststack of cones are about centered around a substantially central openingformed by the truncated apex of one or more of the cones.

Optionally, the cyclone housing may further include a second stack ofcones. In one embodiment, the second stack of cones occupies at least aportion of the second conical portion. In another embodiment, the secondstack of cones occupies at least a portion of the cylindrical portion.In one embodiment, each cone of the second stack of cones is transverseto the cones of the first stack of cones.

In one embodiment, at least one cone in the first stack of cones iscomposed of a metal or a plastic. In another embodiment, at least onecone in the first stack of cones is composed at least partially ofstainless steel. In still another embodiment, at least one cone in thefirst stack of cones is composed entirely of a plastic. Additionally, oralternatively, at least one surface of a cone in the first stack ofcones is coated with a plastic or silicone.

Optionally, an angle of inclination for a surface of a cone in the firststack of cones is between about 30 degrees to about 60 degrees fromvertical. In another embodiment, the angle of inclination of the conesis about 45 degrees.

In another embodiment, each cones of the second stack of cones includesa truncated apex oriented towards the second outlet port. Each cone ofthe second stack of cones may also include an open base oriented towardsthe first outlet port. In one embodiment, the cones of the second stackof cones are generally centered in the cyclone housing. In anotherembodiment, the cones of the second stack of cones are about centeredaround a substantially central opening formed by the truncated apex ofone or more of the cones. When present, the cones of the second stack ofcones may be comprised of at least one of a metal and a plastic. In oneembodiment, at least one cone in the second stack of cones is composedat least partially of stainless steel. In still another embodiment, atleast one cone in the second stack of cones is composed entirely of aplastic. Additionally, or alternatively, at least one surface of a conein the second stack of cones may be coated with a plastic or silicone.

In one embodiment, an angle of inclination for a surface of a cone inthe second stack of cones is between about 30 degrees to about 60degrees from vertical. In another embodiment, the angle of inclinationof the cones in the second stack of cones is about 45 degrees.

In one embodiment, the cones of the first stack of cones have asubstantially uniform spacing. Additionally, the cones of the secondstack of cones may have a substantially uniform spacing. In oneembodiment, the cones of the first stack of cones have a differentspacing compared to the cones of the second stack of cones.

In one embodiment, the at least one inlet is configured as an inlet portin liquid communication with the outside and the inside of the cyclonehousing. In another embodiment, the at least one inlet is associatedwith at least one of the first conical portion, the second conicalportion, and the cylindrical portion of the cyclone housing. In oneembodiment, a first inlet of the at least one inlets is associated withthe cylindrical portion of the cyclone housing. In another embodiment, asecond inlet of the at least one inlets is associated with one of thefirst and second conical portions. In yet another embodiment, the secondinlet is associate with the second conical portion. In anotherembodiment, the at least one inlet is configured to be interconnected toa disposable bioreactor bag. The disposable bioreactor bag may comprisea plastic material.

In one embodiment, the cyclone housing further includes a fluid jacket.The fluid jacket is associated with one or more of the first conicalportion, the second conical portion, and the cylindrical portion. In oneembodiment, the fluid jacket is associated with the second conicalportion and the cylindrical portion. The fluid jacket may include atleast one port to receive a fluid of a predetermined temperature.Optionally, the fluid jacket may include a second port to extract fluidfrom the fluid jacket.

In one embodiment, the cyclone housing further includes a sensor. Thesensor is configured to measure a condition within the cyclone housing.In one embodiment, the sensor comprises a fluorescent probe. Optionally,the sensor is positioned to measure a condition within the secondconical portion. In another embodiment, the sensor is associated with aline interconnected to the second outlet port. In one embodiment, atleast a portion of cyclone housing proximate to the sensor istransparent or translucent. Additionally, or alternatively, the secondconical portion is transparent or translucent. In one embodiment, thesensor comprises a plurality of sensors. In another embodiment, thesensor is operable to measure at least one of pH, dissolved oxygen (DO),Glucose, temperature, and dissolved CO₂(pCO₂). In one embodiment, datafrom the sensor may be used to adjust a temperature of fluid within thefluid jacket. In another embodiment, the data from the sensor may beused to adjust one or more of pH, temperature, dissolved oxygenconcentration, dissolved carbon dioxide, and nutrient concentrationswithin the particle settling device.

It is another aspect of the present disclosure to provide a method ofsettling particles in a suspension. The method includes, but is notlimited to, one or more of: (1) introducing a liquid suspension ofparticles into a particle settling device; (2) collecting a clarifiedliquid from a first outlet port of the particle settling device; and (3)collecting a concentrated liquid suspension from a second outlet port ofthe particle settling device. The particle settling device may be anyparticle settling device disclosed herein.

In one embodiment, the clarified liquid may include a first subset ofparticles of the suspension. The first subset of particles may comprisecell debris, dead cells, and the like.

In one embodiment, the concentrated liquid may include a second subsetof particles of the suspension. The second subset of particles mayinclude live cells. In another embodiment, particles of the secondsubset of particles are generally larger than particles of the firstsubset of particles. In still another embodiment, each particle of thesecond subset of particles generally has a greater mass than theparticles of the first subset of particles.

In one embodiment, the particle settle device includes a cyclonehousing, comprising: (a) a first conical portion; (b) a second conicalportion; (c) a cylindrical portion located between the first and secondconical portions; (d) at least one inlet for the liquid suspension toenter the cyclone housing; (e) the first outlet port for harvesting theclarified liquid; (f) the second outlet port for discharging theconcentrated liquid suspension; and (g) a first stack of cones locatedwithin the cyclone housing. In one embodiment, the first stack of conesoccupies at least part of the first conical portion. Additionally, thefirst stack of cones may occupy at least part of the cylindricalportion.

In one embodiment, each cone of the first stack of cones includes (i) atruncated apex positioned distal to the second conical portion, and (ii)an open base positioned proximate to the second conical portion.Optionally, the cones of the first stack are generally centered around asubstantially central opening formed by the truncated apex in each conein the first stack of cones.

In one embodiment, at least one cone in the first stack of cones iscomposed of a metal or a plastic. In another embodiment, at least onecone in the first stack of cones is composed at least partially ofstainless steel. In still another embodiment, at least one cone in thefirst stack of cones is composed entirely of a plastic. Additionally, oralternatively, at least one surface of a cone in the first stack ofcones is coated with a plastic or silicone.

Optionally, an angle of inclination for a surface of a cone in the firststack of cones is between about 30 degrees to about 60 degrees fromvertical. In another embodiment, the angle of inclination of the conesis about 45 degrees.

In one embodiment, an angle of inclination for a surface of a cone inthe second stack of cones is between about 30 degrees to about 60degrees from vertical. In another embodiment, the angle of inclinationof the cones in the second stack of cones is about 45 degrees. In oneembodiment, the cones of the first stack of cones have a substantiallyuniform spacing.

In one embodiment, the particle settling device further includes a fluidjacket. The fluid jacket is associated with one or more of the firstconical portion, the second conical portion, and the cylindricalportion. In one embodiment, the fluid jacket is associated with thesecond conical portion and the cylindrical portion. The fluid jacket mayinclude at least one port to receive a fluid of a predeterminedtemperature. Optionally, the fluid jacket may include a second port toextract fluid from the fluid jacket.

In another embodiment, the particle settling device further includes asensor. The sensor is configured to measure a condition within theparticle settling device. Optionally, the sensor is positioned tomeasure a condition within the second conical portion. In anotherembodiment, the sensor is associated with a line interconnected to thesecond outlet port. In one embodiment, the sensor comprises a pluralityof sensors. In one embodiment, the sensor is operable to measure atleast one of pH, DO, Glucose, temperature, and pCO₂. In anotherembodiment, the sensor is a fluorescent probe. In one embodiment, atleast a portion of cyclone housing proximate to the fluorescent probe istransparent or translucent. Additionally, or alternatively, the secondconical portion may be transparent or translucent.

Optionally, the method further comprises collecting data from thesensor. Additionally, the method may include using data received fromthe sensor to adjust one or more of pH, temperature, dissolved oxygenconcentration, dissolved carbon dioxide, and nutrient concentrationswithin the particle settling device.

In one embodiment, the liquid suspension comprises at least one of arecombinant cell suspension, an alcoholic fermentation, a suspension ofsolid catalyst particles, a municipal waste water, and industrial wastewater. In another embodiment, the liquid suspension comprises at leastone of mammalian cells, bacterial cells, yeast cells, and plant cells.In another embodiment, the liquid suspension comprises at least one ofalgae cells, plant cells, mammalian and/or murine hybridoma cells (i.e.,cells in different stages of differentiation), stem cells, CAR-T cells,red blood precursor and mature cells, cardiomyocytes or other attachmentprone cells growing attached on microcarrier beads, yeast in beer, andeukaryotic cells. In still another embodiment, the liquid suspensioncomprises recombinant microbial cells selected from at least one ofPichia pastoris, Saccharomyces cerevisiae, Kluyveromyces lactis,Aspergillus niger, Escherichia coli, Bacillus subtilis, and othermicrobial cells. In yet another embodiment, the liquid suspensioncomprises non-cellular particles. For example, the liquid suspension mayinclude one or more of microcarrier beads for attached stem cell growth,an affinity ligand coated microspheric bead or resin, surface activatedmicrospherical beads, and the like.

Optionally, the clarified liquid collected may comprise at least one ofbiological molecules, organic or inorganic compounds, chemicalreactants, and chemical reaction products. In one embodiment, theclarified liquid collected comprises at least one of hydrocarbons,polypeptides, proteins, alcohols, fatty acids, hormones, carbohydrates,antibodies, glycoproteins, terpenes, isoprenoids, polyprenoids,fragrance and flavor compounds, and beer. In another embodiment, theclarified liquid collected comprises at least one of biodiesel, insulinor its analogs, brazzein, antibodies, growth factors, colony stimulatingfactors, and erythropoietin (EPO).

In one embodiment, introducing the liquid suspension into the particlesettling device comprises directing the liquid suspension from a plasticdisposable bioreactor bag into the particle settling device.

Yet another aspect of the present disclosure is a particle settlingdevice, comprising: (A) a cyclone housing; (B) at least two conicalplates disposed inside the cyclone housing; (C) a first opening in thecyclone housing; and (D) a second opening in the cyclone housing. In oneembodiment, the at least two conical plates are stacked one above theother. In one embodiment, the cyclone housing include between about 3and about 30 conical plates.

In another embodiment, the at least two conical plates are separated bya substantially constant distance. Optionally, a width of a channelformed between adjacent surfaces of the at least two conical plates isbetween about 1 mm and about 50 mm. In one embodiment, at least threesupports hold each of the conical plates in the stack.

In one embodiment. each of the conical plates include a truncated apexproximate to the first opening and an open base positioned distal to thefirst opening. The conical plates may be generally centered in thecyclone housing. In one embodiment, the stack of at least two conicalplates is arranged in a substantially vertical position within thecyclone housing. Optionally, an angle of inclination for a surface ofeach of the at least two conical plates is between about 30 degrees andabout 60 degrees from vertical.

Optionally, the at least two conical plates comprise at least onematerial selected from the group consisting of a metal and a plastic. Inone embodiment, the at least two conical plates are comprised stainlesssteel. In another embodiment, at least one surface of the cyclonehousing and the at least two conical plates is coated with a non-stickyplastic or silicone.

In one embodiment, the cyclone housing is comprised entirely of plastic.In another embodiment, the cyclone housing is comprised entirely ofstainless steel.

In one embodiment, the cyclone housing includes a cylindrical portionand a conical portion. In one embodiment, the first opening isassociated with the conical portion. Optionally, the second opening maybe associated with the cylindrical portion. In one embodiment, thesecond opening is positioned in a sidewall of the cylindrical portion.In another embodiment, the second opening is positioned in a lidassociated with an open end of the cylindrical portion. In yet anotherembodiment, the second opening is positioned in the conical portion.

The preceding is a simplified summary of the disclosure to provide anunderstanding of some aspects of the settler devices of this disclosure.This summary is neither an extensive nor exhaustive overview of thedisclosure and its various aspects, embodiments, and configurations. Itis intended neither to identify key or critical elements of thedisclosure nor to delineate the scope of the disclosure but to presentselected concepts of the disclosure in a simplified form as anintroduction to the more detailed description presented below. As willbe appreciated, other aspects, embodiments, and configurations of thedisclosure are possible utilizing, alone or in combination, one or moreof the features set forth above or described in detail below. Additionalaspects of the present disclosure will become more readily apparent fromthe Description of Embodiments, particularly when taken together withthe drawings.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A shows a cross sectional view through the side of one embodimentof a conical spiral settler device of this disclosure;

FIG. 1B is a cross sectional view through the top of the conical spiralsettler device of FIG. 1A, showing a top view of spiral plates inside acyclone housing;

FIG. 2 shows a cross sectional view through another embodiment of thesettler device of this disclosure, without the conical spiral surface;

FIG. 3 shows a cross sectional view of an alternate configuration of aspiral conical surface, with extensions to the conical settler surfaceto ensure the upward flow of cell culture broth through all the conicalspiral and vertical sedimentation chambers within a settler device ofthis disclosure;

FIG. 4 shows a cross sectional view through the side of one embodimentof a conical settler device of this disclosure;

FIGS. 5-7 show schematic diagrams of different liquid flow patternsthrough a bioreactor of this disclosure;

FIG. 8 shows a sectional view through a concentric cylindrical inclinedsettler device of this disclosure, including a vertical sight glass onthe outer surface to show the inclined settling ramps in the outermostannular region;

FIG. 9 shows a top view through a settler device of this disclosure,showing numerous inclined settling ramps welded to inner cylinders inthe annular regions;

FIG. 10 is a sectional view through a settler device of this disclosure,including a first and second stack of cones arranged in oppositeorientations, apex to base, inside a cyclone housing;

FIG. 11 is a sectional view of a settler device of this disclosure,including at least one cone from the first stack of cones that is nottruncated at the apex;

FIG. 12A is a sectional view of another configuration of a settlerdevice of this disclosure in which the top and bottom stack of coneshave different separation distances between successive cones in eachstack;

FIG. 12B is a sectional view of another configuration of a settlerdevice of this disclosure that may function as a cell retention deviceattached to a perfusion bioreactor;

FIG. 13 is a sectional view of another configuration of a settler deviceof this disclosure including only a first stack of cones supported on atleast one rod;

FIG. 14A is a sectional view of another configuration of a settlerdevice of this disclosure including only a first stack of substantiallyequal-sized cones supported on at least one rod;

FIG. 14B is a sectional view of a settler device of an embodiment ofthis disclosure similar to the settler device of FIG. 5A but whichincludes at least one sensor within the lower conical portion of thecyclone housing;

FIG. 14C is a section view of a line configured to be interconnected toa settler device of the present disclosure, the line including at leastone sensor;

FIG. 15 is a schematic representation of the attachment of a compactcell/particle settler device of this disclosure to a modular bioreactor;

FIG. 16 is a graph which shows results of perfusion bioreactor cultureof yeast P. pastoris cells, with a fully packed compact cell settler asthe cell retention device and set up as depicted in FIG. 15;

FIG. 17 shows particle size analysis of samples taken from thebioreactor and settler effluent from the apparatus set up as depicted inFIG. 15;

FIG. 18 shows centrifuge vials containing samples of effluent from thesettler device (tube labeled ‘D’), and from within the bioreactor (tubelabeled ‘C’), and, following centrifugation, cell pellets from effluentfrom the settler device (tube labeled ‘B’), and cells pelleted fromwithin the bioreactor (tube labeled ‘A’);

FIG. 19 is a graph of total protein concentrations in bioreactor andsettler effluent and cumulative harvested protein;

FIG. 20 is a graph of Chinese hamster ovary (CHO) cell perfusionbioreactor 1 Liter interconnected to a 4″ compact cell settler of thepresent disclosure;

FIG. 21 is a histogram of cell/particle sizes of samples taken on day 5from a bioreactor configured as shown in FIG. 15; and

FIG. 22 is another histogram of cell/particle sizes of samples taken onday 5 from a top port of a cell settler device of the present disclosureinterconnected to a profusion bioreactor as shown in FIG. 15.

DESCRIPTION OF EMBODIMENTS

The term “a” or “an” entity refers to one or more of that entity. Assuch, the terms “a” (or “an”), “one or more” and “at least one” can beused interchangeably herein. It is also to be noted that the terms“comprising”, “including”, and “having” can be used interchangeably.

The phrases “at least one,” “one or more,” and “and/or” are open-endedexpressions that are both conjunctive and disjunctive in operation. Forexample, each of the expressions “at least one of A, B and C”, “at leastone of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B,or C” and “A, B, and/or C” means A alone, B alone, C alone, A and Btogether, A and C together, B and C together, or A, B and C together.

The transitional term “comprising” is synonymous with “including,”“containing,” or “characterized by,” is inclusive or open-ended and doesnot exclude additional, unrecited elements or method steps.

The transitional phrase “consisting of” excludes any element, step, oringredient not specified in the claim, but does not exclude additionalcomponents or steps that are unrelated to the disclosure such asimpurities ordinarily associated therewith.

The transitional phrase “consisting essentially of” limits the scope ofa claim to the specified materials or steps and those that do notmaterially affect the basic and novel characteristic(s) of the claimedinvention.

In one embodiment, depicted in FIGS. 1A and 1B, a settler device of thisdisclosure includes a cyclone housing (1) enclosing a spiral verticalplate (7). The spiral vertical plate (7) is joined at one end with aconical surface (8) which tapers down to an opening (9). As depicted inFIG. 1B, the spiral vertical plate (7) is supported within the cyclonehousing by attachment to the cyclone housing (1). Optionally, the spiralvertical plate (7) may also include one or more supporting attachmentsto a top plate (3).

Opening (9) is of sufficient diameter to allow removal of settled cellsor particles. In one embodiment, there is a substantially constantspacing between successive rings of the spiral vertical plate (7). Theconical surface (8) joined to the spiral vertical plate (7) may beformed as a single continuous spiral surface, or individual angledplates, and acts as a lamellar inclined settler plate, in a conicalgeometry.

The cyclone housing (1) optionally includes a means to control thetemperature of the settler device, such as a temperature control jacketor reservoir for cooling or heating fluids to be circulated around allor part of the cyclone housing (1).

The conical bottom portion (2) of the cyclone housing (1) extends from avertical surface of the cyclone housing (1) to the opening (9) and ispreferably positioned at an angle α from the vertical that substantiallymatches the angle of at least one conical surface (8).

The top plate (3), which may function as a lid to the cyclone housing,may be optionally attached to the top of the cyclone housing (1) by atleast one screw (5). The top plate (3) may optionally be secured inplace over the cyclone housing (1) over an o-ring (not shown).

The top port (4) may act as an inlet or outlet port for liquid and/orparticles entering or exiting the settler device through the top plate(3). In one embodiment, the top port (4) is substantially centered inthe top plate (3).

One or more tangential ports (6) located in the cyclone housing (1) mayalso act as one or more of an inlet and outlet port for liquid and/orparticles entering or exiting the settler device through the cyclonehousing (1). These one or more tangential ports (6) may be positioned inthe cyclone housing (1) at any position between the opening (9) and thetop plate (3). In one embodiment, at least one tangential port (6) ispositioned in the conical bottom portion (2). The tangential ports (6)may each be dedicated inlet ports, dedicated outlet ports, or dualfunction inlet/outlet ports, for the transfer of liquid and/or particlesinto or out of the settler device. As noted above, in one embodiment,there is no plug or other impediment preventing the flow of liquid orsuspended particles from the spiral vertical plate (7) or the conicalsurfaces (8), toward the opening (9). Alternatively, a plug may beprovided for one or more of the tangential ports (6) and opening (9).

A modified version of the settler device of this disclosure is depictedin FIG. 2. In this embodiment, the conical surface (illustrated in FIG.1A, reference number 8) is omitted. Thus, the conical bottom portion (2)of the settler device is at least substantially empty. The settlerdevice of the embodiment illustrated in FIG. 2 also works well for manyseparation applications due to the synergistic effects of centrifugalforces acting on particles in this settler device, when a liquidsuspension of particles is introduced through a tangential port (6) nearthe top of cyclone housing (1) proximate the top plate (3) and theincreased residence time of particles in the vertical sedimentationchannel of the vertical conical plate (7).

Another embodiment of the settler device of this disclosure is depictedin FIG. 3. This embodiment is particularly useful for smaller scaleparticle separation applications, such as particle settlers for use witha plastic bag bioreactor using only two vertical ports. In thisembodiment, the settler device of this disclosure includes a cyclonehousing (21) enclosing spiral vertical plate (27). The spiral verticalplate (27) is joined at one end with a conical surface (28) taperingdown to an opening (29) in a conical bottom portion (22). The spiralvertical plate (27) is supported within the cyclone housing (21) byattachment to the cyclone housing (not shown). Optionally, the spiralvertical plate (27) may also include one or more supporting attachmentsto a top plate (23). The cyclone housing (21), including the conicalbottom portion (22), of this embodiment may also include a means tocontrol the temperature of the settler device, such as a fluid jacket asdescribed herein.

The spiral vertical plate (27) is formed with progressively longervertical spirals, moving from the center of the settler device of thisembodiment towards the cyclone housing (21). The conical surfaces (28)joining one end of the spiral vertical plate (27) are formed inincreasingly longer lengths to extend from the joined end of the spiralvertical plates (27) to a position proximate the center of the settlerdevice, in order to direct cells or particles towards the opening (29).

As depicted in FIG. 3, the end of the conical surfaces (28) opposite theend joining the spiral vertical plate (27) may extend beyond the centerof the settler device to partially overlap successive conical surfaces(28). In one embodiment, there is no plug or other impediment preventingthe flow of liquid or suspended particles from the spiral vertical plate(27) or the conical surfaces (28), toward the opening (29). As with theembodiment depicted in FIG. 1, in one embodiment there is asubstantially constant spacing between successive rings of the spiralvertical plate (27). Optionally, the conical surface (28) joined to thespiral vertical plate (27) may be formed as a single continuous spiralsurface, or individual angled plates. For use in small scale, bioreactoror biobag separation applications, opening (29) may function for inletof cell culture broth as well as recycling settled cells or particlesback to a biobag or bioreactor. Top plate (23) is optionally attached tothe top of the cyclone housing (21) by at least one screw (25), and maybe secured in place over the cyclone housing (21) over an o-ring (notshown). Top port (24) may act as an inlet or outlet port for liquidand/or particles entering or exiting the settler device through the topplate (23). In this embodiment, top port (24) is particularly useful forremoving clarified cell culture liquid. In one embodiment, top port (24)is substantially centered in the top plate (23).

Similarly, one or more optional tangential ports (26) located in thecyclone housing (21) and/or the conical bottom portion (22) may also actas an inlet or outlet port for liquid and/or particles entering orexiting the settler device through the cyclone housing (21). These oneor more optional tangential ports (26) may be positioned in the cyclonehousing (21) and/or the conical bottom portion (22) at any positionbetween the opening (29) and the top plate (23). The optional tangentialports (26) may each be dedicated inlet ports, dedicated outlet ports, ordual function inlet/outlet ports, for the transfer of liquid and/orparticles into or out of the settler device. Such optional tangentialport (26) located in the cyclone housing (21) proximate the top plate(23) is typically not needed in small scale, bioreactor or biobagseparation applications, but may be useful for faster filling of thesettler device with cell culture liquids before priming a pump in liquidcommunication with the central top port (24), as described below. If theoptional tangential inlet port (26) is not used, the cell culture brothcan be sucked up through opening (29) by a peristaltic pump in fluidcommunication with the top port (24), as described below.

Another embodiment of the settler device of this disclosure is depictedin FIG. 4. This embodiment is particularly useful for separationapplications in which the particle settling device requires regular orcontinual service, such as disassembly and cleaning of the conicalsettling surfaces within the settler device. In this embodiment, thesettler device of this disclosure includes a cyclone housing (31)enclosing a stack of two or more stacked cones (32), each having acentral opening (33), the cyclone housing (31) tapering down to anopening (39).

The stacked cones (32) comprise at least three vertical supports (34)supporting each cone (32) above the next successive cone (32) in thestack. In preferred embodiments, the vertical supports (34) arepreferably placed at about a constant distance and are formed at asubstantially equal length to hold each successive cone (32) in thestack at substantially an equal spacing between all of the cones (32) inthe stack. There should be at least three vertical supports for eachcone (32) to properly support each cone, but each cone (32) may comprisemore than three vertical supports as needed to adequately or properlysupport the cone (32). However, each vertical support represents animpediment to settled particles or cells sliding down the surface of thecone (32) towards the central opening (33).

The vertical supports (34) may be attached to the top of each cone (32),thereby supporting the next successive cone (32) in the stack.Alternatively or additionally, the vertical supports (34) may beattached to the bottom of each cone (32), thereby supporting the cone(32) above the next successive cone (32) in the stack.

Optionally, in another embodiment, the vertical supports (34) maycomprise at least three L-shaped spacers or triangular spacersinterconnected to an upper surface of each cone (34) that is distal tothe truncated apex of the cone. The L-shaped spacers include a firstside interconnected to a second side at an apex. In one embodiment, theL-shaped spacers and/or triangular spacers are interconnected to theupper surface such that the first side supports the next successive cone(32) in the stack. The second side is substantially parallel to thesurface of the cone (32). Optionally, the second side may project beyondthe cone (32) to space the cone a predetermined distance from aninterior surface of the cyclone housing (31). In one embodiment, theL-shaped spacers and the triangular spacers have a substantially thincross-section to prevent or minimize interference with the movement orflow of liquid and suspended particles within the cyclone housing (31).Optionally, the L-shaped spacers and/or the triangular spacers may beintegrally formed with the cone (32). In another embodiment, theL-shaped spacers and/or the triangular spacers are interconnected to thecone (32).

In one embodiment, there is no plug or other impediment preventing theflow of liquid or suspended particles from the central opening (33) ineach cone (32) toward the opening (39).

As depicted in FIG. 4, the cyclone housing (31), may include a means tocontrol the temperature of the settler device, such as a jacket orreservoir (35) for cooling or heating fluids to be circulated around allor part of the cyclone housing (31). Ports (36, 37) may be inlet oroutlet ports for the circulation of heating or cooling fluids throughthe reservoir (35).

Top plate (38) is optionally attached to the top of the cyclone housing(31) by at least one screw (42), and may be secured in place over thecyclone housing (31) over an o-ring (not shown). Top port (40) may actas an inlet or outlet port for liquid and/or particles entering orexiting the settler device through the top plate (38). Top port (40) isparticularly useful for removing clarified cell culture liquid. In oneembodiment, the top port (40) is substantially centered in the top plate38.

Similarly, one or more optional tangential ports (41) located in thecyclone housing (31) may also act as an inlet or outlet port for liquidand/or particles entering or exiting the settler device through thecyclone housing (31). These one or more optional tangential ports (41)may be positioned in the cyclone housing (31) at any position betweenthe opening (39) and the top plate (38). The optional tangential ports(41) may each be dedicated inlet ports, dedicated outlet ports, or dualfunction inlet/outlet ports, for the transfer of liquid and/or particlesinto or out of the settler device.

In each of the embodiments of this disclosure, the number of spirals orcones typically range from about 3 to about 30 or more, depending on theradius of the device. In each of the embodiments of this disclosure, thechannel width (i.e., the distance between each successive spiral or eachsuccessive conical cone) can range between about 1 mm and to about 50mm. For larger flow rates, device sizes, and dense fluids, the largerchannel width will be preferable to minimize the pressure drop orfriction. A smaller channel width can increase the number of spirals orcones that can fit inside a given radius of the device. Smaller channelwidths are, however, more prone to clogging by dense packing of thesettled or settling particles. The thickness of spiral or cone materialshould be as small as possible to maintain the rigidity of shape whileminimizing the weight of the spiral or cones supported inside thecyclone housing.

The radius and size of these settler devices can be scaled up easily inthree dimensions, as much as needed for large-scale/large-volumeprocesses. However, the scale up of these devices needs to be carriedout empirically, as theoretical development of predictive equations isnot yet available, as they were for lamellar settlers (Batt et al.1990). These settler devices can be scaled up or down to suit theseparation needs of different industries or applications or sizes as theseparation surface is scaled up or down approximately in threedimensions, compared to the more typical one- or two-dimensional scalingof previous settling devices.

In each of the embodiments of this disclosure, the angle of inclinationof the surfaces of the conical surfaces or the stacked cones can also bebetween about 30 degrees and about 60 degrees from the vertical. Incertain embodiments, the angle of inclination for the surfaces of theconical surfaces or stacked cones is about 45 degrees from the vertical.As described above, for the separation of stickier particles (typicallymammalian cells), the angle of inclination is preferably closer to thevertical (i.e., about 30 degrees from the vertical). For less-stickysolid particles (for example, catalyst particles), the angle ofinclination can be further from the vertical (preferably, about 60degrees from vertical).

The material of construction of any of the settler devices of thisdisclosure can be stainless steel (especially stainless steel 316), orsimilar materials used for applications in microbial or mammalian cellculture, as well as other metals used for applications in chemicalprocess industries, such as catalyst separation and recycle. In certainembodiments, the settler devices of this disclosure include stainlesssteel surfaces that are partially or completely electropolished toprovide smooth surfaces that cells or particles may slide down aftersettling out of liquid suspension. In certain embodiments, some or allof the surfaces of the settler device may be coated with a non-stickyplastic or silicone, such as dimethyldichlorosilane. In relatedembodiments, the material construction of any of these settler devicesmay be non-metals, including plastics, for use in, for example, singleuse disposable bioreactor bags, etc. While metal settling devices of thedisclosure can be constructed via standard plate rolling and welding ofsteel angular plates to the bottom of the spiral plate, a plasticsettler device of this disclosure, or individual parts thereof, may bemore easily fabricated continuously as a single piece using, forexample, injection molding or three-dimensional printing technologies.

In each of the embodiments of this disclosure, liquid may be directedinto, or drawn out of, any of the ports or openings in the conicalsettling device by one or more pumps (for example a peristaltic pump) inliquid communication with the port or opening. Such pumps, or othermeans causing the liquid to flow into or out of the settler devices, mayoperate continuously or intermittently. If operated intermittently,during the period when the pump is off, settling of particles or cellsoccurs while the surrounding fluid is still. This allows those particlesor cells that have already settled to slide down the inclined conicalsurfaces unhindered by the upward flow of liquid. Intermittent operationhas the advantage that it can improve the speed at which the cells slidedownwardly, thereby improving cell viability and productivity. In aspecific embodiment, a pump is used to direct a liquid suspension ofcells from a bioreactor or fermentation media into the settler devicesof the present disclosure.

In each of the embodiments of this disclosure, the top plate, or lid,covering the cyclone housing may be concave, rising to a central corepoint. In these embodiments, the angle of rise in the concave top platemay preferably be between about 1 degree and about 10 degrees, morepreferably between about 1 degree and about 5 degrees. This concave topplate creates a tent-like space above the center of the cyclone housing.Gas, bubbles, froth or the like may accumulate in this space and a tubemay be inserted through an opening in the cyclone housing or through anopening in the top plate to withdraw such gasses, etc. from the spacebeneath the top of the cyclone housing. Similarly, fluid or gas may bepumped into the cyclone housing through such tube that is insertedthrough an opening in the cyclone housing or through an opening in thetop plate.

Methods of Use and Operation Processes

The settling devices of this disclosure have applications in numerousfields, including: (i) high cell density biological (mammalian,microbial, plant or algal) cell cultures secreting polypeptides,hormones, proteins or glycoproteins, sub-unit vaccines, viruses,virus-like particles or other small chemical products, such as ethanol,isobutanol, isoprenoids, etc.; (ii) separating and recycling porous ornon-porous solid catalyst particles catalyzing chemical reactions inliquid or gas phase surrounding solid particles; (iii) separating andcollecting newly formed solids in physical transformations such ascrystallization, flocculation, agglomeration, precipitation, etc., fromthe surround liquid phase; and (iv) clarifying process water in largescale municipal or commercial waste water treatment plants by settlingand removing complex biological consortia or activated sludge or othersolid particles.

FIG. 5 shows an effective flow pattern of liquid and particles through asettler device of this disclosure, producing maximal particle separationefficiency. As depicted in FIG. 5, a particle containing liquid(including, for example, cell culture liquid, waste water or reactionfluid containing solid catalyst particles, etc.) is preferablyintroduced tangentially into a settler device of this disclosure nearthe top of the cylindrical housing from the side along the direction ofarrow (51), to take full advantage of the centrifugal forces on theparticles, pushing them against the wall of the spiral vertical plate.The channel within the spiral vertical plate creates increased contactarea, residence time and gradually increasing centrifugal force for theparticles to be pushed against the spiral wall. The particles or cellssliding down the walls and settling in the vertical sedimentationcolumns of the spiral channel enter an enhanced sedimentation zone ofthe conical surfaces. Particles or cells settled on the inclined conicalsurfaces are swept down to the opening at the bottom of the conicalhousing by the dense liquid (i.e. liquid containing concentratedparticles or cells) exiting at the bottom of the cone in the directionof arrow (53). Liquid exiting the outlet in the direction of arrow (53)contains concentrated cells or particles to be recycled to a bioreactoror directed to a chemical reactor, or waste water tank, etc. Clarifiedliquid containing any secreted proteins or other products and smallerparticles or dead cells or cell debris, is harvested at an outlet alongthe direction of arrow (52).

In one embodiment, clarified liquid entering the central tube is removedor harvested at the top by suction from a pump attached on the tubeconnected to the top port. The dense liquid containing concentratedparticles or cells can be recycled to the reactor or bioreactor orharvested as desired. The flow rate of the dense liquid exiting thebottom of the conical device is ideally equal to the difference in theinlet flow rate at the tangential entry near the top and outlet flowrate at the top, each controlled by a separate pump. Additional controlvalves may be added to the bottom liquid exit tube to ensure that theclarified liquid exits at the top and may be fully opened as needed toprevent or remove any dense packing of particles clogging the underflowstream.

Another flow configuration for liquid and particles through a settlerdevice of this disclosure is depicted in FIG. 6. This flow configurationresults in a slightly reduced separation efficiency compared to the flowconfiguration depicted in FIG. 5 because the top vertical entry does nottake advantage of any small centrifugal forces which can be created bythe tangential entry depicted in FIG. 5. Nevertheless, thisconfiguration makes use of the major separating principle of enhancedsedimentation on inclined surfaces and will be sufficient for fullseparation of larger live mammalian cells from smaller dead cells andcell debris if the device is sized adequately for use with a bioreactor.

In this operating embodiment, liquid containing cells or solidparticles, or waste water is directed into the top of the settler devicealong the direction of arrow (61). Outlet liquid containing concentratedcells, particles or sludge to be recycled back to the bioreactor,chemical reactor or waste water tank exits the settler device along thedirection of arrow (62). Clarified liquid containing any secretedproteins, smaller dead cells or cell debris, is harvested from thesettler device near the top of the conical housing proximate the top ofthe settler device, along the direction of arrow (63).

A third flow configuration useful for a settler device of thisdisclosure that includes only two ports is depicted in FIG. 7. A liquidsuspension is directed along the direction of arrow (72) from asingle-use disposable plastic bioreactor bag (71), which may beculturing either mammalian or microbial cells secreting one or moredesired chemical products, into a bottom port of the settler device. Theinlet port is firmly attached to the plastic bioreactor bag (71), butwithout any pump. This inlet port carries both the contents of thebioreactor bag upwards, and the settled cells downward back to thebioreactor bag. Thus, the feed inlet to the settler device and theunderflow of settled particles or cells cross paths in the same bottomport of the conical settler device, i.e., the two streams (feed inletand underflow) occur via the same bottom port. This flow configurationmay be useful in connection with a single use, plastic disposablebioreactor bag, or with other applications used with smaller scalesettler devices of this disclosure. Such smaller scale settler devicesare typically made of plastic, and may be single-use, disposable plasticdevices. In this flow configuration, clarified liquid outlet containingany secreted protein product and fewer smaller cells or cell debris,exits from the top port of the settler device along the direction ofarrow (73).

If a third port is provided in the configuration of FIG. 7, it may beused initially to provide a vacuum suction to fill up or prime thedevice. In some embodiments, the third port is not provided in thesettler device as it is not needed in conjunction with this embodimentcontaining a single port in which feed inlet and underflow of settledparticles or cells cross paths in the same bottom port.

For the smaller scale applications with a plastic bag bioreactor withonly two vertical ports used in the flow configuration as shown in FIG.7, it is advantageous to extend the conical spiral surface closer to thecenter of the settler device to prevent a direct flow of inlet cellculture broth from the bottom port up through the central opening in thedevice. One possible extension of the conical spiral surface to ensurethe flow of cell culture broth from bottom inlet through all the spiralconical and vertical sedimentation chambers of the device is shown inthe sectional diagram of FIG. 3.

Referring to FIG. 8, a sectional view of a concentric cylindricalinclined settler device of this disclosure includes an outer wall (81)of cylindrical section of the cyclone assembly, shown in FIG. 8 with anoptional fluid jacket (92), a conical portion (82) of the cycloneassembly, with the optional fluid jacket (92) extending to this conicalportion (82), a lid (83) on top of the assembly, a tangential port inlet(84) for a liquid (for example, a cell culture), entering near the topof conical portion (82), through the optional fluid jacket (92), abottom outlet port (85) for returning concentrated liquid (for example,a concentrated cell culture containing settled cells to a bioreactor),and a top outlet port (86) for harvesting the clarified liquid (forexample, culture liquid containing very few cells, which are mostlysmaller dead cells and cell debris).

Concentric cylindrical tubes (87) are located within the outer wall(81). Annular strips (not shown) are attached to the concentriccylindrical tubes (87) at an angle between about 30 degrees to about 60degrees (or, in another embodiment, about 45 degrees) from vertical. Inone embodiment, the annular strips are attached to the inner cylinder,but not to the outer cylinder. Concentric cones (88) channel settledparticles (for example, cells) to the bottom outlet port (85).

A first fluid port (89) accesses the optional fluid jacket (92) on theoutside of the cyclone assembly. A second fluid port (90) accesses theoptional fluid jacket (92) near the top of the cylindrical section (81)of the cyclone assembly. The first fluid port (89) may be used to inject(or remove) a fluid of a predetermined temperature into the optionalfluid jacket (92). The second fluid port (90) may be used to remove (orinject) the fluid from the optional fluid jacket (92). In this manner, aselected fluid may circulate at a predetermined rate through the fluidjacket (92). Accordingly, the fluid jacket (92) may be used to heat orcool the cyclone assembly or maintain a predetermined temperature of thecyclone assembly. In one embodiment, the fluid for the fluid jacket (92)comprises water; however, other fluids are contemplated for use with thecyclone assemblies of the present disclosure.

As depicted in FIG. 8, an optional sight glass (91) is provided showingthe inclined settler strips attached to the inside cylinder in theoutermost annular region between the cylindrical tubes. As noted above,in one embodiment of the present disclosure, annular strips are notattached to the outer cylinder, intentionally leaving a small(approximately 0.5 mm-10 mm) gap between the strips and the outercylinder, thereby allowing the settled particles to fall down throughthis gap.

As depicted in FIG. 8, the settler devices of this disclosure mayinclude a closure or lid (83) over at least a portion of the settlerdevice at an end of the settler device opposite the bottom outlet port(85). The closure or lid (83) may also include an outlet or port (86)for removing gases and liquids or entering liquids into the settlerdevice. The opening and the additional ports or outlets in the lid arein liquid communication with the outside and the inside of the settlerdevice to allow the passage of liquids and gases into and/or out of thesettler device, and in each instance of such opening or inlet/outlet,these passageways into and out of the cyclone housing may include valvesor other mechanisms that can be opened or closed to stop or restrict theflow of liquids into or out of the settler devices of this disclosure.

The lid (83) covering the settler device may be concave, rising to acentral core point. The angle of rise in the concave top plate maypreferably be between about 1 degree and about 10 degrees, moreoptionally between about 1 degree and about 5 degrees. Such concave topplate creates a tent-like space above the center of the settler device.Gas, bubbles, froth or the like may accumulate in this space and a tubemay be inserted through an opening in the settler device or through anopening in the top plate to withdraw such gasses, etc. from the spacebeneath the top of the settler device. Similarly, fluid or gas may bepumped into the settler device through such tube that is insertedthrough an opening in the settler device or through an opening in thelid. As depicted in FIG. 9, a top view of the concentric cylindricalinclined settler device of this disclosure shows numerous annular stripsattached to the outside of each cylinder (87). The strips may beattached to the vertical cylinders at an angle between about 30 degreesto about 60 degrees to the vertical (typically at an angle of about 45degrees to the vertical). As shown in FIG. 9, small (approximately 1 mm)spacings (93) are provided between each inclined settler strip and thenext successive outer cylinder of each annual region, to allow thesettled particles to fall down along the outer cylindrical wall onto theconcentric cones in the bottom section of the assembly.

These settler devices may include a means to control the temperature ofthe settler device, such as reservoir for cooling or heating fluids tobe circulated around all or part of the outer wall of the settlerdevice. Ports may be inlet or outlet ports for the circulation ofheating or cooling fluids through the reservoir.

A lid is optionally attached to the top of the settler device by one ormore screws, and may be secured in place over the settler device over ano-ring.

Methods of Use and Operation of Processes of the Settling DevicesDepicted in FIGS. 8 and 9

Referring now to the settling device depicted in FIGS. 8 and 9 of thisdisclosure, exemplary methods of using the settling devices aredescribed.

A particle containing liquid (including, for example, cell cultureliquid, waste water or reaction fluid containing solid catalystparticles, etc.) is introduced tangentially into a device of thisdisclosure though the port (84) near the top of the conical section (82)of cyclone housing assembly. Approximately 50%-99% of the enteringliquid (typically about 90%) is removed through the bottom port (85),while the remaining 1%-50% (typically about 10%) of the liquid isremoved through the top port (86). A pump (such as a peristaltic pump)may be used to suck liquid out of this top port (86), while theconcentrated liquid exiting the bottom may be allowed to exit the bottomoutlet (85) of the cyclone housing due to gravity, without the need fora pump. Most of the entering cells (or particles) are pushed against theconical walls of this assembly (88) through centrifugal forces uponentry, settle down the conical portion through a gentle vortex motioninitially, getting faster as the liquid and particles/cells go down andexit via the bottom port. The rest of the cells, which have not settled,will move up through the annual regions in between the numerous inclinedsettling strips attached to the inside cylinder. As the liquid movesslowly up the annular inclined channels, bigger particles (e.g., livecells) will settle on the ramp and either slide down the ramp or morelikely fall down the small (approximately 1 mm) spacing provided betweenthe ramps and the outer walls of each annular region. These settledparticles fall down vertically along the outer cylindrical walls untilthey reach the bottom conical section of the assembly and proceed toslide down the conical section to the bottom port (85).

By increasing the liquid flow rate through top port (86), it is possibleto reduce the residence time of liquid inside the inclined settlingzones such that smaller particles (for example dead cells and cellulardebris) will not have settled by the time the liquid reaches the top ofthe settling zone, and therefore these smaller particles exit thesettling device via the top port (86). This feature provides a simplemethod to remove smaller particles (such as dead cells and cellulardebris) selectively via the top port (86) into a harvest stream, whilelarger particles (such as live and productive cells) are returned fromthe bottom port (85) to another vessel (such as a bioreactor).

Thus, in these methods, the step of introducing a liquid suspension intothe settler device includes directing a liquid suspension from a plasticbioreactor bag into the particle settling device.

Liquid may be directed into, or drawn out of, any of the ports oropenings in the settling device by one or more pumps (for example aperistaltic pump) in liquid communication with the port or opening. Suchpumps, or other means causing the liquid to flow into or out of thesettler devices, may operate continuously or intermittently. If operatedintermittently, during the period when the pump is off, settling ofparticles or cells occurs while the surrounding fluid is still. Thisallows those particles or cells that have already settled to slide downthe inclined conical surfaces unhindered by the upward flow of liquid.Intermittent operation has the advantage that it can improve the speedat which the cells slide downwardly, thereby improving cell viabilityand productivity. In a specific embodiment, a pump is used to direct aliquid suspension of cells from a bioreactor or fermentation media intothe settler devices of the present disclosure.

In certain embodiments of these methods, the clarified liquid collectedfrom the settler device includes at least one of biological molecules,organic or inorganic compounds, chemical reactants, and chemicalreaction products. In certain embodiments of these methods, theclarified liquid collected from the settler device includes at least oneof hydrocarbons, polypeptides, proteins, alcohols, fatty acids,hormones, carbohydrates, antibodies, isoprenoids, biodiesel, and beer.In certain embodiments of these methods, the clarified liquid collectedfrom the settler device includes at least one of insulin or its analogs,monoclonal antibodies, growth factors, sub-unit vaccines, viruses,virus-like particles, colony stimulating factors and erythropoietin(EPO).

Referring now to FIG. 10, which is a cross-sectional view of acylindrical inclined settler device of this disclosure, an embodiment ofthe settling devices of this disclosure includes: an outer wall (100) ofa cyclone housing (101), which includes an optional fluid jacket (102),a first, upper conical portion (103) of the cyclone housing (101), asecond, lower conical portion (104) of the cyclone housing (101), avertical (or generally cylindrical) portion (108) of the cyclone housing(101), located between the first, upper conical portion (103) and thesecond, lower conical portion (104), with the optional fluid jacket(102) extending to conical portions (103) and/or (104), and verticalportion (108), of the cyclone housing, an inlet (105) for a liquid (forexample, cell culture media) entering the cyclone housing (101), whichinlet (105) extends through the optional fluid jacket (102), a bottomoutlet port (106) for returning concentrated liquid (for example, aconcentrated cell culture liquid containing settled cells) back to abioreactor or other vessels such as harvest or collection or holdingtanks, and a top outlet port (107) for harvesting a clarified liquid(for example, culture liquid containing very few cells, which are mostlysmaller dead cells and cell debris).

A first stack of cones (109) is located within the outer wall (100) ofthe cyclone housing, occupying the first, upper conical portion (103)and at least part of the vertical portion (108) of the cyclone housing(101). The first stack of cones (109) is generally centered around asubstantially central opening (110) in the first stack of cones (109).Each of the cones in the first stack of cones (109) illustrated in FIG.10, are truncated cones (i.e., the shape is “truncoconical”) as the apexof each cone is truncated to form an opening that, in conjunction withthe other cones in the first stack of cones (109), forms the centralopening (110). As depicted in FIG. 10, the cones (109) comprising thefirst stack of cones (109) are arranged within the outer wall (100) ofthe cyclone housing (101) with the truncated apex of each cone (109)oriented towards the top outlet port (107) and the open base of the coneoriented towards the bottom outlet port (106).

A second stack of cones (111) is optionally located within the outerwall (100) of the cyclone housing (101), occupying the second, lowerconical portion (104) and at least part of the vertical portion (108) ofthe cyclone housing (108) and generally centered around the centralopening (110) in the second stack of cones (111). The central opening(110) extends from the first stack of cones (109). Similarly, each ofthe cones in the second stack of cones (111) illustrated in FIG. 10, aretruncated cones, and the apex of each cone is truncated to form anopening that, in conjunction with the other cones in the second stack ofcones (111), continues the central opening (110). As depicted in FIG.10, the cones comprising the second stack of cones (111) are arrangedwithin the outer wall (100) of the cyclone housing with the open base ofeach cone oriented towards the top outlet port (107) and the truncatedapex of the cone oriented towards the bottom outlet port (106), (i.e.,the open bases of the first stack of cones (109) is oriented in theopposite direction of the second stack of cones (111)). In oneembodiment, the opening (110B) formed by the truncated apex of each cone(111) in the second stack of cones has about the same diameter as theopening (110A) formed by the truncated apex of each cone (109) of thefirst stack of cones.

As illustrated in FIG. 10, at least one of the cones in the first stackof cones (109) is attached to a cone in the second stack of cones (111).Such attachment is typically proximate an end of both the cone in thefirst stack of cones (109) and the cone in the second stack of cones(111), opposite the central opening (110). In one embodiment, theattachment may be configured to form an extension or overlap (112) ofthe at least one cone in the first stack of cones (109) at the point ofattachment to the cone in the second stack of cones (111).

The cones comprising the first stack of cones (109) and the second stackof cones (111), may include a projection (113) supporting the nextsuccessive cone in the stack. These projections (113) are preferablyplaced at a constant distance and are formed at an equal size to holdeach successive cone in the stack at about an equal spacing between allof the cones in the stacks. At least three projections (113) are neededfor each cone to properly support each successive cone, but each conemay comprise more than three projections (113), as needed to adequatelyor properly support the cone (only two such projections (113) areillustrated in the cross-sectional view of FIG. 10). For example, eachcone may comprise between four and eight projections (113), or maycomprise more than eight projections (113), to support the nextsuccessive cone in the stack. The projections (113) are attached to atleast one surface of a cone, but these projections do not attach toanother cone in a stack of cones. Thus, these projections do not attachtwo or more cones in a stack of cones to one another. The projections(113) extend from at least one surface of a cone to support the nextsuccessive cone in the stack, or to support the cone above the nextsuccessive cone in the stack (i.e., the projections may extend above acone, or extend below a cone, or extend both above and below a cone).The projections are typically of uniform size such that the projectionssupport each cone in the first and second stack of cones (109 and 111)at a substantially uniform distance between each cone in the stack.

The projections may be configured as pins (114) that extend from asurface of a cone to support each successive cone in a stack of cones.Such pins may project at an angle between about 30 degrees to about 120degrees from the surface of the cone. In a preferred configuration,illustrated in FIG. 10, the pins (114) project both above and below thesurface of at least one cone in a stack of cones, at an angle of about90 degrees from the surface of the cone, to support the next successivecone in the stack, and to support the cone above the next successivecone in the stack.

In another embodiment, the pins (114) may optionally have an “L” shapeor a triangular shape. The L-shaped and/or triangular spacers (114) maybe interconnected to an upper surface of each cone (109) that is distalto port 106. The L-shaped spacers (114) include a first sideinterconnected to a second side at an apex. In one embodiment, theL-shaped spacers (114) and/or triangular spacers (114) areinterconnected to the upper surface such that the first side supportsthe next successive cone (109) in the stack. The second side issubstantially parallel to the surface of the cone (109). Optionally, thesecond side may project beyond the cone (109) to space the first stackof cones (109) a predetermined distance from an interior surface of thewall (100) of cyclone housing (101). In one embodiment, the L-shapedspacers and the triangular spacers (114) have a substantially thincross-section to prevent or minimize interference with the movement orflow of liquid and suspended particles within the cyclone housing (101).Optionally, the L-shaped spacers and/or the triangular spacers may beintegrally formed with the cones (109). In another embodiment, theL-shaped spacers and/or the triangular spacers are separately formed andsubsequently interconnected to the cones (109).

A cylindrical inclined settler device of this disclosure may optionallyinclude at least one spacer configured to prevent a stack of conesresiding within the outer wall (100) of the cyclone housing fromshifting to touch the interior walls of the cyclone housing. Asillustrated in FIG. 10, spacer (115) extends along the end of the firststack of cones (109) opposite the central opening (110), andapproximately parallel to the outer wall (100) of the cyclone housing.In this configuration, the spacer (115) prevents the first stack ofcones (109) from shifting or falling against the vertical portion (108)of the cyclone housing, for example if the cyclone housing (101) is seton its side or inverted while the stacks of cones remain inside thecyclone housing. The spacer (115) may be formed as a ring that encirclesthe circumference of the cyclone housing with three or more “legs” thatextend substantially parallel to the vertical portion of the cyclonehousing (108), thereby supporting the stacks of cones on several sides.

The spacer (115), formed as a ring that encircles the circumference ofthe cyclone housing as described above, may be attached to at least onerod at a first point of attachment. The at least one rod extendssubstantially parallel to the vertical portion of the cyclone housing(108), horizontally across the cyclone housing, and again substantiallyparallel to the vertical portion of the cyclone housing (108) to attachto the spacer (115) at a second point of attachment, on a sidesubstantially opposite the first point of attachment. This spacer (115),attached to at least one rod is shown in the cross-sectional view of thesettler devices depicted in FIGS. 12A, 12B, 13, 14A, 14B, 14C (137, 144,151, 159 respectively). These rods may support an upper, first stack ofcones independently from the lower, second stack of cones.

As depicted in FIGS. 13, 14A, 14B, and 14C, this configuration allowsthe settler devices of this disclosure to be constructed and used withonly an upper, first stack of cones (109), i.e., without a lower, secondstack of cones below the rod (151 and 159). In these configurations, thelower conical portion (104) of the cyclone housing (152 and 160) may actas a receptacle or incubator for live cells. Thus, in thisconfiguration, port (145 and 153) may function to supply liquids and/orgasses, as needed to support the growth of cells. Thus, such chemicalsas cellular growth nutrients, pH modifying chemicals and buffers, andoxygen, nitrogen, or carbon dioxide may be added or removed from port(145 and 153). Similarly, bottom outlet port (146 and 154), may alsofunction as an inlet or outlet port for the introduction and/or removalof such liquids or gasses.

The settler devices illustrated in FIGS. 10-14C may include a means tocontrol the temperature of the settler device, such as a reservoir forcooling or heating fluids to be circulated around all or part of theouter wall of the settler device. Thus, an optional fluid jacket (102)may be included on the outside of the cyclone housing. Water or otherfluids may be directed into the fluid jacket (102) to maintain thecyclone housing and all of its contents within a desired temperaturerange. Ports may be formed in the outer wall (100) of the cyclonehousing, to reach the jacket (102). The ports may function as inlet oroutlet ports for the circulation of cooling or heating fluids throughthe jacket.

As depicted in FIG. 10, the settler devices of this disclosure mayinclude a closure or lid (116) over at least one end of the settlerdevice opposite the bottom outlet port (106). The closure (116) may alsoinclude a port (107) for removing liquids from, or entering liquidsinto, the settler device. The top outlet port (107) and any additionalports or outlets in the lid are in liquid communication with the outsideand the inside of the settler device to allow the passage ofliquids/gases into and/or out of the settler device, and in eachinstance of such opening or inlet/outlet, these passage ways into andout of the cyclone housing may include valves or other mechanisms thatcan be opened or closed to stop or restrict the flow of liquids/gasesinto or out of the settler devices of this disclosure.

As depicted in FIG. 10, the lid (116) covering the settler device may beconcave, rising to a central core point. The angle of rise in theconcave lid (116) may be between about 20 degrees and about 60 degrees.In another embodiment, the concave lid (116) rises at an angle ofbetween about 30 degrees and about 50 degrees. Gas, bubbles, froth orthe like may accumulate in this space and a tube may be attached to anopening in the settler device or through top outlet port (107) towithdraw such gasses, etc. from the space beneath the top of the settlerdevice. Similarly, fluid or gas may be pumped into the settler devicethrough such tube that is inserted through an opening in the settlerdevice or through an opening in the lid (116). The lid (116) may beattached to the settler device by one or more screws, and may be securedin place over the settler device over an o-ring.

As depicted in FIG. 11, one or more cones (120) in the first stack ofcones (109) located within the outer wall (100) of the cyclone housing,occupying the first, upper conical portion (103) and at least part ofthe vertical (or cylindrical) portion (108) of the cyclone housing, andgenerally centered around a central opening (110) in the first stack ofcones (109), may be devoid of a central opening, or may have a muchsmaller central opening than other cones in this first stack of cones.These cones (120) may direct particles or cells settling within thecentral opening (110) to the surfaces of cones in the first stack ofcones (109). These cones (120) may also direct particles or cellssettling within the central opening (110) to the surfaces of conesresiding in the second stack of cones (111). Similar to the settlerdevice depicted in FIG. 10, second stack of cones (111) is arranged withthe truncated apex of each cone oriented towards the bottom outlet port(106) and the open base of the cone oriented towards the top outlet port(107)). In one embodiment of the cyclone housings of FIGS. 10, 11,spacing between the cones of the first stack of cones (109) issubstantially the same as the spacing between the cones of the secondstack of cones (111). Optionally, in another embodiment cyclone housingsof FIGS. 10, 11, the space between the cones of the first stack of cones(109) is one of greater than and less than the spacing between the conesof the second stack of cones (111).

FIGS. 12A and 12B depict another configuration of a settler device ofthis disclosure. The devices illustrated of FIGS. 12A and 12B, whichdepict cross-sectional views of a cell or particle retention device thatmay be attached to a perfusion bioreactor, include: an inlet (131 and138) of cell culture liquid that may be pumped in continuously from theperfusion bioreactor, an outlet port (132 and 139) from whichconcentrated settled cells flow out and are returned to a bioreactor, atop outlet port (133 and 140) from which clarified culture fluid flows,which fluid may contain a secreted product, as well as some of thenot-yet settled smaller dead cells and cellular debris, an optionalsecond inlet (134 and 141) from the bioreactor, which inlet may pass gasand/or cellular culture media, a first port (135 and 142) forcooling/heating fluid to an optional fluid jacket, and a second port(136 and 143) to the fluid jacket. In one embodiment, the first port(135, 142) is an inlet into the optional fluid jacket and the secondport (136, 143) is an outlet from the optional fluid jacket.

In these examples of a settler device of this disclosure, cell cultureliquid from a bioreactor is pumped into the settler via the tangentialinlet port (131 and 138) near the top of the bottom conical section(104). Any gas in the liquid inlet can be easily separated with at-junction on the inlet line and the upper line carrying mostly the gaswith some cell culture liquid can be inlet via the upper tangential port(134 and 141) near the top of the cylindrical portion (108) of thesettlers. The clarified harvest output containing the secreted proteinis harvested continuously from the top outlet (133 and 140) of the cellretention device, while the concentrated cells from the bottom outlet(132 and 139) are recycled back to the bioreactor, resulting in a highcell density perfusion bioreactor that can be operated indefinitely,i.e. over several months of continuous perfusion operation. Thecontinuous high titer harvest from a single, 1000-liter, high celldensity perfusion bioreactor can easily exceed the accumulatedproduction from a large (>20,000 liter) fed-batch bioreactor on anannual basis.

As illustrated in FIG. 12A, the spacing between the cones in the firstand second stacks (109, 111) can be different. This may be advantageous,for example when it may be desirable to have liquid flow be larger inthe bottom section (104), and cell separation in the top or first stackof cones (109) is more efficient with smaller spacing than is typicallyrequired for the bottom stack of cones (111). As illustrated in FIG.12B, the spacing between the cones in the first and second stacks (109,111) can be the same or substantially the same, allowing for similarflow rates for liquid and particles or cells in both stacks of cones.

FIGS. 13, 14A and 14B, depict cross-sectional views of a cell orparticle separation devices that can function as a stand-alone perfusionbioreactor for the in vitro expansion of mammalian cells, such as stemcells and CAR-T cells for autologous cell therapy. In these examples ofsettler devices of this disclosure, inlet of serum-free or animalprotein-free cell culture medium may be pumped continuously into thesettler/perfusion bioreactor, through bottom port (146 and 154) and/orside port (145 and 153). A controlled mixture of O₂, CO₂, and N₂ mayalso be pumped in to control the pH and DO of the culture supernatantinside the settler/bioreactor. At the end of in vitro cell expansion,the concentrated settled cells collecting at the bottom can be harvestedfrom bottom port (146 and 154).

Clarified culture fluid containing any metabolic waste products, such asammonia and lactate, or gasses, along with any not-yet settled smallerdead cells and cell debris, may be removed through top port (147 and155). Optional liquid outlet (148 and 156) from the bioreactor may beused for sampling bioreactor contents, for example to check cellviability, and continuous measurement of liquid pH and DO for inputsinto a computer-controlled multi-gas mass flow controller.Cooling/heating fluid may be directed into (or out of) a first port (149and 157) to the fluid jacket (102) and flow out from (or into) a secondport (150 and 158) to the fluid jacket (102).

FIG. 13 depicts a configuration of the compact cell settler device thatcan function as a stand-alone perfusion bioreactor in which the lowerconical portion (104) of the cyclone housing (152) is empty of a secondstack of cones and may thus house cells in culture. The surface of thecyclone housing at least in the lower conical portion of the cyclonehousing (152) may comprise a plastic, and/or may be coated with aplastic or other material that supports the growth of cells in culture.

FIG. 14A depicts another possible configuration of the compact cellsettler device that can function as a stand-alone perfusion bioreactorin which the lower conical portion (104) of the cyclone housing (160)and at least part of the vertical portion (108) of the cyclone housing(161) are empty of a stack of cones and may thus house cells in culture.The surface of the cyclone housing at least in the lower conical portionof the cyclone housing and the vertical portion of the cyclone housing(160 and 161) may comprise a plastic, and/or may be coated with aplastic or other material that supports the growth of cells in culture.

During operation of the settler devices of the embodiments depicted inFIGS. 13 and 14A, inlet of serum-free or animal protein-free cellculture medium is pumped continuously into the settler/perfusionbioreactor, through bottom port (146, 154) and/or side port (145, 153).A controlled mixture of O₂, CO₂, and N₂ may also be pumped in to controlthe pH and DO of the culture supernatant inside the settler/bioreactor.At the end of in vitro cell expansion, the concentrated settled cellscollecting at the bottom can be harvested from bottom port (146, 154).Clarified culture fluid containing any metabolic waste products, such asammonia and lactate, or gasses, along with any not-yet settled smallerdead cells and cell debris, may be removed through top port (147, 155).Optional liquid outlet (148, 156) from the bioreactor may be used forsampling bioreactor contents, for example to check cell viability, andcontinuous measurement of liquid pH and DO for inputs into acomputer-controlled multi-gas mass flow controller.

FIG. 14B depicts another compact cell settler device of the presentdisclosure which is similar to the device of FIG. 14A. The cell settlerdevice may be used as a stand-along bioreactor/cell sorter combination.Accordingly, the cell settler device of the embodiment of FIG. 14B maybe used without the perfusion bioreactor described in conjunction withFIG. 15.

Sensors (170) are positioned within the cyclone housing (160). In oneembodiment, the sensors (170) are arranged on an interior surface (164)of the cyclone housing (160). As describe above, the surface (164) ofthe cyclone housing at least in the lower conical portion (104) maycomprise a plastic. In one embodiment, the plastic is transparent or atleast translucent. Optionally, at least a portion of the cyclone housing(160) is transparent or translucent. For example, a portion (165) oftransparent or translucent material may be interconnected to an aperturein the surface (164) of the cyclone housing similar to a window. Thetransparent portion (165) may comprise glass, plastic, or any othersuitable material. The transparent portion (165) may be formed of amaterial which is transparent to light of a predetermined range orranges of wavelengths.

The sensors (170) are in contact with media within the cyclone housing(160). Each sensor (170) is operable to monitor one or more of pH, DO,Glucose, temperature, and CO₂ (including dissolved or partial CO₂) inthe cyclone housing (160). Growth media may be added to the cyclonehousing (160) through side port (153). In this manner, the embodiment ofthe compact cell settler device illustrated in FIG. 14B may be used as abioreactor without an external modular bioreactor such as illustrated inFIG. 15.

In one embodiment, each sensor (170) measures one of pH, DO, Glucose,temperature, and CO₂. Optionally, one or more of the sensors maycomprise a fluorescent probe. As one of skill in the art willappreciate, the fluorescent probes (170) emit light (171) that variesbased on a condition sensed by the probe. The light (171) passes throughthe surface (164) or the transparent portion (165) and is collected by areader or meter (173). Optionally, the light may be collected by anoptional fiber cable (172) and transmitted to the meter (173). The meter(173) is operable to report or display levels of at least one of pH, DO,Glucose, temperature, and CO₂ sensed by the fluorescent probes (170).

The fluorescent probes (170) may be affixed in a variety of differentpositions within the cyclone housing (160). Thus, the probes (170) canbe arranged to measure different conditions, or changes of conditions,at different areas of the cyclone housing. In one embodiment, the probes(170) are spaced from ports (153, 154) of the cyclone housing (160).

In one embodiment, the stand-along bioreactor/cell sorter combinationillustrated in FIG. 14B can be fabricated in single-use disposableplastic. Alternatively, the stand-along bioreactor/cell sortercombination of the embodiment of FIG. 14B can be manufactured of moredurable stainless steel for larger scale perfusion bioreactors. Like allembodiments of the present disclosure, the device of FIG. 14B may bescaled to any desired size.

Referring now to FIG. 14C, a partial view of a line (217) that may beinterconnected to any of the compact cell settler devices of the presentdisclosure is illustrated. The line (217) may have a diameter orotherwise be configured to interconnect to any port (4, 6, 9, 24, 26,29, 39, 40, 41, 84, 85, 86, 105, 106, 107, 131, 132, 133, 134, 138, 139,140, 141, 145, 146, 147, 148, 153, 154, 155, 156) of embodiments of thepresent disclosure. The line (217) may optionally include at least onesensor (170E, 170F) positioned within a hollow interior. The sensors(170E, 170F) are in contact with fluid and/or particles within the line(217). Optionally, the sensors (170E, 170F) are arranged on an interiorsurface of the line (217) although other configurations arecontemplated.

Sensors (170E, 170F) may be the same as, or similar to, the sensors(170) described in conjunction with FIG. 14B. Accordingly, each sensor(170E, 170F) is operable to monitor one or more of pH, DO, Glucose,temperature, and CO₂ (including dissolved or partial CO₂) in the line(217). In one embodiment, each sensor (170E, 170F) measures one of pH,DO, Glucose, temperature, and CO₂.

Optionally, one or more of the sensors (170E, 170F) may comprise afluorescent probe which emits light (171) that varies based on acondition sensed by the probe (170E, 170F). The light (171) is collectedby a reader or meter (173). Optionally, the light (171) may be collectedby an optional fiber cable (172) and transmitted to the meter (173). Themeter (173) is operable to report or display levels of at least one ofpH, DO, Glucose, temperature, and CO₂ sensed by the fluorescent probes(170E, 170F).

In one embodiment, line (217) may comprise a material that istransparent or at least translucent. Thus, light (171) generated bysensor (170E) may pass through the line (217). In another embodiment, atleast a portion (174) of the line (217) is transparent or translucent,similar to a window. Accordingly, light (171) generated by the sensor(170F) may be transmitted through window portion (174) and collected bymeter (173).

Methods of Use and Operation of Processes Referring now to the settlingdevice depicted in FIGS. 10 and 11 of this disclosure, exemplary methodsof using the settling devices are described. A particle containingliquid (including, for example, cell culture liquid, waste water orreaction fluid containing solid catalyst particles, etc.) is introducedtangentially into a device of this disclosure though the port (105).Approximately 50%-99% of the entering liquid (typically about 90%) isremoved through the bottom port (106), while the remaining 1%-50%(typically about 10%) of the liquid is removed through the top port(107). A pump (such as a peristaltic pump) may be used to suck liquidout of this top port (107), while the concentrated liquid exiting thebottom may be allowed to exit the bottom outlet (106) of the cyclonehousing due to gravity, without the need for a pump. Alternately, theliquid containing the settled cells or particles, may be pumped out fromthe bottom port (106) of the conical settler at about 50%-99% ofentering liquid flow rate, and the remaining clarified liquid (1-50%)may exit via the top port (107). Optionally, fluid exiting port (107)may be pumped out into a harvest line.

Most of the entering cells (or particles) are pushed against the wallsof this assembly (100) through centrifugal forces upon entry, settledown the conical portion through a gentle vortex motion initially,getting faster as the liquid and particles/cells go down and exit viathe bottom port (106). Cells or particles which have not settled willmove up through the stacks of cones (109 and 111). As the liquid movesslowly up through the stacks of cones (109 and 111), bigger particles(e.g., live cells) will settle on the surfaces of the cones and eitherslide down the cones or fall down the small spacing provided between thecones and the outer walls of the cyclone housing (100). These settledparticles fall down vertically along the outer cylindrical walls untilthey reach the bottom conical section of the assembly (104) and proceedto slide down the conical section to the bottom port (106).

By increasing the liquid inlet flow rate through port (105), it ispossible to reduce the residence time of liquid inside the inclinedsettling zones such that smaller particles (for example dead cells andcellular debris) will not have settled by the time the liquid reachesthe top of the settling zone, and therefore these smaller particles exitthe settling device via the top port (107). This feature provides asimple method to remove smaller particles (such as dead cells andcellular debris) selectively via the top port (107) into a harveststream, while larger particles (such as live and productive cells) arereturned from the bottom port (106) to another vessel (such as abioreactor).

Thus, in these methods, the step of introducing a liquid suspension intothese settler devices may include directing a liquid suspension from aplastic bioreactor bag into the particle settling device.

Liquid may be directed into, or drawn out of, any of the ports oropenings (105, 106, 107) in the settling device by one or more pumps(for example a peristaltic pump) in liquid communication with the portor opening. Such pumps, or other means causing the liquid to flow intoor out of the settler devices, may operate continuously orintermittently. If operated intermittently, during the period when thepump is off, settling of particles or cells occurs while the surroundingfluid is still. This allows those particles or cells that have alreadysettled to slide down the inclined conical surfaces unhindered by theupward flow of liquid. Intermittent operation has the advantage that itcan improve the speed at which the cells slide downwardly, therebyimproving cell viability and productivity. In a specific embodiment, apump is used to direct a liquid suspension of cells from a bioreactor orfermentation media into the settler devices of the present disclosure.

One parameter that may be adjusted in these methods of using the settlerdevices of this disclosure is the liquid flow rate into and out of thesettler devices. The liquid flow rate will depend entirely on theparticular application of the device and the rate can be varied in orderto protect the particles being settled and separated from the clarifiedliquid. Specifically, the flow rate may need to be adjusted to protectthe viability of living cells that may be separated in the settlerdevices of this disclosure and returned to a cell culture, but the flowrate should also be adjusted to prevent substantial cell or particlebuild up in the settler devices or clogging of the conduits thattransfer liquid into and out of the settler devices.

In examples of these methods, the clarified liquid collected from thesettler device includes at least one of biological molecules, organic orinorganic compounds, chemical reactants, and chemical reaction products.In certain embodiments of these methods, the clarified liquid collectedfrom the settler device includes at least one of hydrocarbons,polypeptides, proteins, alcohols, fatty acids, hormones, carbohydrates,antibodies, isoprenoids, biodiesel, and beer. In examples of thesemethods, the clarified liquid collected from the settler device includesat least one of insulin or its analogs, monoclonal antibodies, growthfactors, sub-unit vaccines, viruses, virus-like particles, colonystimulating factors and erythropoietin (EPO).

Each publication or patent cited herein is incorporated herein byreference in its entirety. The settling devices of the presentdisclosure now being generally described will be more readily understoodby reference to the following examples, which are included merely forthe purposes of illustration of certain aspects of the embodiments ofthe present disclosure. The examples are not intended to limit thedisclosure, as one of skill in the art would recognize from the aboveteachings and the following examples that other techniques and methodscan satisfy the claims and can be employed without departing from thescope of the present disclosure.

EXAMPLES Example 1: Yeast or Other Microbial Cells Secreting ProteinProducts

Recombinant microbial cells, such as yeast or fungal (Pichia pastoris,Saccharomyces cerevisiae, Kluyveromyces lactis, Aspergillus niger, etc.)or bacterial (Escherichia coli, Bacillus subtilis, etc.) cells, whichhave been engineered to secrete heterologous proteins (for example,insulin or brazzein) or naturally secreting enzymes (e.g. A. niger, B.subtilis, etc.) can be grown in bioreactors attached to the compactsettler device of this disclosure, to recycle live and productive cellsback to the bioreactor, which will thereby achieve high cell densitiesand high productivities. Fresh nutrient media is continuously suppliedto the live and productive cells inside the high cell densitybioreactors and the secreted proteins or enzymes are continuouslyharvested in the clarified outlet from the top port (or top-side outletsas shown in FIGS. 5, 6 and 7), while the concentrated live andproductive cells are returned back to the bioreactor. As dead cells anda small fraction of live cells are continuously removed from thebioreactor via the harvest outlet, cell growth and protein productioncan be maintained indefinitely, without any real need for terminatingthe bioreactor operation. In operations using yeast Pichia cells withthe conical settler devices of this disclosure, the perfusion bioreactorhas been operated for over a month. As the microbial cells grow insuspension culture and the cell retention device can be scaled up to anydesired size, this disclosure can be attached to suspension bioreactorsof sizes varying from lab scale (<1 liter) to industrial scale (>50,000liters) or any size therebetween to achieve high cell density perfusioncultures.

In one specific example, a perfusion bioreactor culture of yeast Pichiapastoris cells is described. Yeast Pichia pastoris cells were grown in a5-liter, computer-controlled bioreactor, initially in batch mode to growthe cells from the inoculum for the first 50 hours, then in fed-batchmode to fill up the attached 12-liter cell settler slowly for the next100 hours, and then in continuous perfusion mode with a compact cellsettler of this disclosure to remove the smaller dead cells and recyclethe larger live cells back into the bioreactor. A typical schematic ofthe attachment of a compact cell/particle settler of this disclosure toany modular bioreactor is shown in FIG. 15.

Referring to FIG. 15, the yeast Pichia pastoris cells were grown in aperfusion bioreactor (218). Growth media was added to the bioreactor(218) from media reservoir (200) via a first pump (202) interconnectedto input line (201). Dissolved oxygen content and pH were continuouslymonitored in the bioreactor (218) by dissolved oxygen monitor (206) andpH monitor (204). Yeast cell culture from the bioreactor (218) wasdelivered to a 12-liter compact cell settler (208) of the presentdisclosure via a second pump (214) interconnect to line (212). Effluentfrom the compact cell settler (208), which contained smaller dead cells,was evacuated by effluent line (210). Larger live cells were recycledfrom the cell settler (208) back to the bioreactor (218) via third pump(216) and return line (217). Media and cell culture levels in thebioreactor (218) were controlled by removing excess cell culture viafourth pump (220) and removal line (222) to be captured or discarded.

Results obtained with this perfusion bioreactor set up with a compactcell/particle settler of this disclosure are shown in FIG. 16. Thecircles show the optical density of bioreactor samples, measured at 600nm, building up during the initial batch and fed-batch culture period ofabout 150 hours, followed by continuous perfusion operation up to 1600hours or longer than 2 months. The settler effluent or harvest rate isadjusted by manipulating either settler inlet pump setting and/orsettler recycle pump setting. The cell concentration (as measured by ODat 600 nm) and the size distribution are determined by the harvest flowrate and cell size distribution of the cells entering from thebioreactor and other factors such as the recycle ratio from the settler.The effluent stream contains very little cells, as measured by the verylow OD's in the range from 0 to 30, even as the perfusion rate isgradually increased from 2000 ml/day to over 6,000 ml/day. These resultsdemonstrate that very high cell density was obtained and maintained inthe bioreactor due to the recycle of most of the live cells back to thebioreactor and selective removal of smaller dead cells and cell debris.Even at these increasing perfusion rates, the bioreactor can be operatedindefinitely at high cell density without any reason to terminate thebioreactor, such as clogged membranes in competing membrane based cellretention devices.

Samples from the bioreactor and settler effluent taken at the same timepoint were analyzed with a particle size analyzer. The normalized cellsize distribution results shown in FIG. 17 clearly indicate that thesettler effluent contains a significantly smaller cell size distributioncompared to that found for the cells in the bioreactor. These resultsdemonstrate that the settler removed the smaller dead cells and any celldebris preferentially in the effluent, while the larger live cells arepreferentially returned to the bioreactor. Thus, the bioreactor iscontinuously cleaned by selective removal of dead cells and cell debrisby the settler effluent and consequently there is no accumulation ofdead cells and cell debris within the bioreactor, as happens routinelywith all other cell retention devices.

The bioreactor and settler effluent samples from an early time pointduring the perfusion culture were collected and centrifuged in small 2ml vials. FIG. 18 shows centrifuge vials containing samples of effluentfrom the settler device (208) (tube labeled ‘D’) and from within thebioreactor (218) (tube labeled ‘C’) and the cell pellets followingcentrifugation: cells pelleted from effluent from the settler device(208) (tube labeled ‘B’) and cells pelleted from within the bioreactor(218) (tube labeled ‘A’). The pelleted cells from the bioreactor occupyalmost 50% of the wet packed cell volume in the vial (tube “A”), whilethe pelleted cells in the settler effluent occupy only about 5% of thewet packed cell volume of tube “B”. These results again confirm thatonly a very small fraction of the intact smaller cells from thebioreactor are removed in settler effluent while most of the largerintact cells are preferentially returned to the bioreactor.

Total protein concentrations in the bioreactor and settler effluentduring this 2 month long perfusion operation were measured and shown inFIG. 19. These results show that after the initial batch and fed-batchoperation, i.e. during the prolonged perfusion operation, total proteincontent in the effluent sample (squares) from the settler device (208)is consistently greater than the total protein content in the samplefrom the bioreactor (218) (triangles). These results suggest verystrongly that there is no protein sieving inside the settler (208), asis commonly observed with membrane-based cell retention devices such asATF in perfusion cultures of mammalian cells. Further these resultssuggest that there is some additional protein production in the settler(208), causing the effluent protein concentrations to be consistentlyhigher than those in the bioreactor (218) at the same time.

The total accumulated protein in the harvest stream (circles) from thecontinuous perfusion bioreactor configuration illustrated in FIG. 15 canbe compared with protein can be harvested in the cell-free supernatantof a single fed-batch bioreactor (218) performed over 158 hours oralmost 6 days, and repeated again and again over the same cultureduration of say 1600 hours. While fed-batch cultures typically have along downtime to harvest or empty the bioreactor, clean the internalsurfaces, sterilize in situ with steam, cool, refill the bioreactor withsterile medium, inoculate the bioreactor with fresh cells and then allowthe cells to grow to high enough cell density to see significantincrease in the protein titer, the continuous perfusion bioreactorcontinues to operate uninterrupted at high cell density and highproduction rate throughout the culture operation. Consequently, thetotal accumulated protein in the continuously harvested product streamis increasing, at a significantly faster rate as the perfusion rate isincreased, and accumulates to 160 g, 5× higher protein amount than canbe harvested in the cell-free supernatants from 8 repeated fed-batchculture operations in the same 5 liter bioreactor.

Example 2: Removing Yeast Cells from Beer

In large-scale brewing operations, yeast cells are removed from theproduct beer by filtration devices, which regularly get clogged, orcentrifugation devices, which are expensive high-speed mechanicaldevices. Previously, hydrocyclones were unsuccessfully tested for thisapplication (Yuan et al., 1996; Cilliers and Harrison, 1997). Thesedevices can be readily replaced by the settler devices of thisdisclosure to clarify beer from the top outlets and remove theconcentrated yeast cell suspension from the bottom outlet. Due to theincreased residence time and enhanced sedimentation in the conicalsettler zones of this disclosure, the inventor has achieved successfulseparation of yeast cells from cell culture liquid, harvesting theculture supernatant containing only about 5% of the cells entering thesettler device in its first operation. As the device can be scaled up ordown to increase or decrease its cell separation efficiency, it isfeasible to obtain completely cell-free beer from the harvest port, ifdesired. Thus, the devices of this disclosure may be particularly usefulin brewing beer, as well as clarifying beer, and in continuous brewingarrangements.

Example 3: Mammalian Cell Perfusion Cultures

Enhanced sedimentation of murine hybridoma and recombinant mammaliancells in inclined settlers have already been demonstrated successfully(Batt et al., 1990 and Searles et al., 1994) and scaled up in lamellarsettlers (Thompson and Wilson, U.S. Pat. No. 5,817,505, 1998). While thelamellar settlers are scaled up in three dimensions independently, aconical settler device of this disclosure can be scaled up in threedimensions simultaneously by simply increasing its radius, as discussedabove. Thus, the settlers of this disclosure are more compact, containmuch more inclined surfaces for settling on a smaller footprint, and aremore easily scalable cell retention devices with proven applications inmammalian cell cultures secreting glycoproteins, such as monoclonalantibodies, and other therapeutic proteins. The clarified harvest outputfrom the top port containing the secreted protein is harvestedcontinuously from the cell retention device, while the concentratedcells from the bottom outlet are recycled back to the bioreactor,resulting in a high cell density perfusion bioreactor, that can beoperated indefinitely, (i.e. over several months of continuous perfusionoperation). The continuous high titer harvest from a single, 1000-liter,high cell density perfusion bioreactor can be more than the accumulatedproduction from a large (>20,000 liter) fed-batch bioreactor on anannual basis.

Recombinant Chinese hamster ovary cells, which are used commonly in theoverexpression and secretion of therapeutic glycoproteins, are culturedin a 1-liter controlled bioreactor attached with a 4″ compact cellsettler (FIG. 14A), as shown schematically in FIG. 15. Viable celldensities in the bioreactor (circles), settler top effluent (triangles)and settler bottom return to the bioreactor (squares) are shown in FIG.20. Soon after the perfusion operation starts at 60 hours, we can seethat very few live cells are removed from the settler top effluent andincreasing amount of viable cells are being returned to the bioreactorfrom the settler bottom outlet. Consequently the bioreactor viable celldensity (VCD) is increasing gradually after the perfusion operationbegins and more dramatically the viability percentage (diamonds) in thebioreactor increases when the perfusion begins.

Cell size distributions were measured on samples from the bioreactor andsettler top effluent on day 5 and shown in FIGS. 21 and 22 respectively.FIG. 21 represents a histogram of cell/particle sizes measured by aBeckman-Coulter Multisize Analyzer for the bioreactor sample, showing abroad distribution of live cells and possibly doublets in sizes rangingfrom about 10 microns to about 30 microns with a peak of about 16microns, a sharp peak of dead cells in sizes between 8 and 9 microns andhuge tail of cell debris in the smaller size range smaller than 8microns.

FIG. 22 represents another histrogram of cell/particle sized measured bythe same instrument on the sample from the top port effluent of thecompact cell settler (208), showing an enhanced peak of dead cells insize between 8 and 9 microns, a tail of cell debris in the sizes smallerthan 8 microns and dramatically a total absence of any peak for livecells about 16 microns. These size measurements strongly demonstratethat settler top effluent removes selectively the smaller dead cells andcell debris from the perfusion bioreactor (218), while the larger livecells are continuously returned to the perfusion bioreactor (218). Thisselective removal of smaller dead cells and cell debris has beendemonstrated by us (Batt et al. 1990 and Searles et al. 1994) withinclined plate settlers. The present disclosure of compact cell settlersagain reproduced those successive results in a more compact and moreeasily scalable design. None of the other cell retention devicesavailable today for mammalian cells exhibit any such selectivity inremoving only the smaller dead cells and cell debris.

Example 4: Vaccines, Viruses or Virus-Like Particles Production

Production of vaccines, such as viruses or virus-like particles (VLPs),is usually carried out by infection and lysis of live mammalian orinsect cells in a batch or fed-batch bioreactor culture. Viruses orvirus-like particles are released from the infected cell in a lyticprocess after large intracellular production of these viruses orvirus-like particles. With the large difference in the size (sub-micronor nanometer scale) of these particles compared to the size (about 5-20microns) of live mammalian and insect cells, the separation of theviruses or virus-like particles from the bioreactor culture is verysimple. By controlling the harvest or outlet rate of cell culture brothcontaining mostly viruses or VLPs, along with cell debris, it ispossible to retain a smaller number of the infective particles insidethe bioreactor along with the growing live cells to continually infectand produce vaccines in a continuous perfusion bioreactor attached to asettler device of this disclosure for continuous harvest of viruses andVLPs.

Example 5: Solid Catalyst Particle Separation and Recycle

Separation of a solid catalyst particle for recycle into the reactor andreuse in further catalyzing liquid phase chemical reactions, such asFischer-Tropsch synthesis, has been demonstrated before with lamellarsettlers (U.S. Pat. No. 6,720,358, 2001). Many such two-phase chemicalreactions, involving solid catalyst particles in liquid or gas phasereactions can be enhanced by the particle settling devices of thisdisclosure, which presents a more compact particle separation device toaccomplish the same solids separation and recycle as demonstrated withlamellar settlers.

Example 6: Plant and Algal Cell Harvesting

Recombinant plant cell cultures secreting valuable products, while notyet commercially viable, are yet another field of potential applicationsfor the settling devices of this disclosure. Inclined settlers have beenused in several plant cell culture applications. Such devices can bereplaced by the more compact conical spiral settler devices of thisdisclosure. With the size of plant cells being much higher than those ofyeast or mammalian cells, the cell separation efficiency will be muchhigher with single plant cells or plant tissue cultures.

A more immediate commercial application of the settler devices of thisdisclosure may be in the harvesting of algal cells from large scalecultivation ponds to harvest biodiesel products from inside algal cells.Relatively dilute algal cell mass in large (acre sized) shallow pondsconverting solar energy into intracellular fat or fatty acid storage canbe harvested easily through the conical spiral settler device of thisdisclosure, and the concentrated algal cells can be harvested from thebottom outlet.

Example 7: Municipal Waste Water Treatment

Large scale municipal waste water treatment plants (using activatedsludge or consortia of multiple bacterial species for degradation ofbiological and organic waste in sewage or waste water) commonly uselarge settling tanks and more modern versions of these plants uselamellar settlers to remove the clarified water from the sludge. Theconical spiral settler devices of this disclosure can be scaled up tothe larger sizes required in these plants, while remaining smaller insize than the large settling tanks or lamellar settlers currently usedin these treatment plants.

Example 8: Industrial Process Water Clarification

Large scale water treatment plants, cleaning either industrial wastewater or natural sources of turbid water containing suspended solids,use large scale settling tanks or lamellar inclined settlers. Theselarge scale devices can now be replaced with the more compact conicalspiral settler devices of this disclosure to accomplish the same goal ofclarifying water for industrial reuse or municipal supply of freshwater.

Example 9: Capture and Purification of Monoclonal Antibodies on Proteina Coated Beads

Cell culture supernatants containing monoclonal antibodies can becontacted with protein A coated microspheres or beads (40-200 microns)inside our settler via two different inlets, e.g. beads coming in from atop inlet and the cell culture supernatant coming in via the bottom portto maximizing their contacting and capture efficiency. Capture ofmonoclonal antibodies on protein A beads is very quick, typically under10 min. of residence time inside the competing affinity chromatographycolumns. The protein A coated microspheric beads will settle down fastand can be kept in suspension and well mixed contact with the cellculture supernatant by pumping it in from the bottom inlet. The depletedcell culture supernatants can be removed continuously from the topoutlet of cell settler (208 of the present disclosure in a batch loadingoperation. Any beads entrained with upward flowing liquid will settle onthe inclined surfaces and return to the bottom stirred region. Afterloading close to the maximum binding capacity of the add beads, beadscan be washed with the typical washing solution of about 3-5× volume ofthe settler to remove all unbound host cell protein along with deadcells cell debris which are present in the supernatant via the topoutlet.

After completing thorough washing, elution media will be pumped inslowly to remove the bound antibodies into the liquid medium andconcentrated antibody solution is removed via the top port, whileretaining the beads inside the settler. After elution is completed,equilibration of the beads is conducted by pumping in the equilibrationsolution from the bottom inlet, while the beads are held in suspensionby this incoming solution. After equilibration, next batch of cellculture supernatant is loaded into the settler to repeat the abovefour-step process, similar to the sequence used in a chromatographycolumn. Some advantages of using the cell settler devices of the presentdisclosure for monoclonal antibody capture are that: (i) cell culturesupernatant can be directly loaded to contact with the protein A beads,without the need for removing dead cells or cell debris commonly presentin the supernatant; and (ii) more efficient immediate contacting of allthe suspended beads with in the incoming supernatant, rather than thegradual or delayed exposure of monoclonal antibodies to the fixed bed ofbeads in the later parts of the column.

Example 10: Decanter/Cell Settler for In Situ Extraction of SecretedOrganic Products from Yeast or any Other Cells into an Organic Layer

Production and secretion of several fragrance and flavor compounds arebeing metabolically engineered into microbial yeast cells, such asSaccharomyces cerevisiae. Some of these compounds may be more toxic tothe cells and can be extracted readily into an organic liquid to reducethe cellular toxicity as well as to increase the productivity of theyeast cells. Emulsions of organic liquid containing the secreted productand aqueous layer containing the productive microbial cells from thestirred tank bioreactor can be pumped into the inlet port (131) of thecompact cell settler. Inside the quiet zones of the settler, theemulsion is separated easily into the organic layer floating on top andharvested via the top port (133) and aqueous layer containing the liveand productive cells settling to the bottom and recycled to thebioreactor via bottom port (132). Any cellular debris will fractionateinto the organic layer and easily removed from the top of settler. Liveand productive cells in the aqueous layers are returned to thebioreactor to increase the cell densities and productivity inside theperfusion bioreactor.

Example 11: In Vitro Expansion of Various Mammalian Cells, Such as StemCells and CAR-T Cells for Autologous Cell Therapy in Our Compact CellSettler that can be Used as a Stand-Alone Perfusion Bioreactor

Currently, the field of in vitro expansion of various mammalian cellssuch as stem cells and CAR-T cells is expanding rapidly with sterilesingle-use disposable culture bags as the bioreactors placed on rockingplatform for mixing or inside a CO₂ incubator for pH control. Such bagbioreactors are increasingly operated in continuous perfusion mode toremove the accumulated waste metabolic by-products, such as ammonia andlactate, using microfiltration membranes as cell retention devices onthe bag to maintain high cell viability during the expansion. However,during the prolonged perfusion operation, dead cells and cell debrisaccumulate in these bags and cannot be removed through themicrofiltration membranes on the bag. The cell settler devices of thisdisclosure can be operated effectively as a stand-alone, air-liftbioreactors, operated in a continuous perfusion to bring in freshnutrient and remove metabolic waste products, as well as to removeselectively any dead cells and cell debris. The bottom port can be usedas an inlet for controlled mixture of multiple gases CO₂, O₂ and N₂ tomaintain the desired pH and DO in the bioreactor. The rising air throughthe central portion entrains or carries up some cell culture liquid,provides a gentle mixing of the nutrients in the bioreactor, and exitsat the top outlet, while the liquid is disengaged in the cylindricalportion of settler and is recycled over the conical settlers. Thereturning cell culture liquid can be sampled for continuous measurementsof pH, DO, for inputs into computer controlling the inlet gas mixtureand occasional sampling for cell density and viability as desired. Afterthe desired cell expansion, concentrated live cells are collected viathe bottom port by switching the gas flow to a cell collection bag. Themajor advantage of our cell settler/bioreactor is that it provides for afacile removal of dead cells and cell debris along with toxic metabolicwaste by-products, resulting in a high cell density of live cells afterin vitro expansion for autologous cell therapy.

Example 12: Continuous Separation of Precipitated and ConcentratedTherapeutic Proteins

Several therapeutic proteins (e.g. insulin analog glargine andmonoclonal antibodies) can be precipitated by adding simple salts (e.g.zinc chloride for glargine, or ammonium sulfate for antibodies),adjusting pH, and other solvents (e.g. m-cresol or other phenolics forglargine and ethanol for antibodies). This precipitation is a low-costalternative to chromatography in the downstream purification processesfor these therapeutic proteins. Currently, these precipitation steps arecarried out in the batch mode, followed by centrifugation or decantationto remove the supernatant from the precipitant.

Using the separation devices of the present disclosure, a continuousseparation process may be implemented. The protein rich harvest medium(after removing any cells by micro filtration or centrifugation or othermethods) is input into a compact cell settler of this disclosure, alongwith other required chemicals, such as solvents, or salts in apH-modifying solution, such as NaOH or HCl. The precipitation processwill occur inside the settler and the protein-rich precipitant can becontinuously removed in the bottom outlet, away from theprotein-depleted supernatant, which is removed continuously from the topoutlet.

Example 13: Ex Vivo Expansion of Mesenchymal Stromal/Stem Cells (MSCs)on Microcarrier Beads and Purification of Expanded Stem Cells

MSCs are capable of ex vivo expansion in the presence of suitable growthmedium and are commonly grown attached to surfaces, such as tissueculture flasks, petri dishes, roller bottles, cell cubes, andmicrocarrier beads. Attached growth on microcarrier beads (size rangingfrom 100 microns to 500 microns) is very easily scalable as they aresuspended in stirred or agitated bioreactors, controlled for optimalgrowth conditions such as pH, temperature, dissolved oxygenconcentration and nutrient concentrations. However, separation ofexpanded stem cells from the microcarriers is a challenge, requiringenzymatic detachment, washing off excess enzyme quickly, and separatingthe stem cells from microcarrier beads. These different steps arecurrently attempted using labor-intensive and contamination-prone batchprocessing steps. Each of these difficult steps can be accomplished moreeasily in the bioreactor/cell sorter device shown in FIG. 14B whichincludes sensor probes (170) positioned within the cyclone housing(160). In one embodiment, the sensor probes comprise fluorescent probesto measure one or more of pH, dissolved oxygen (DO), glucoseconcentrations, temperature, and CO₂ levels within the cyclone housing.More specifically, with the settler device illustrated in FIG. 14B: (i)the excess enzyme is very easily washed or removed via the top port(155) by feeding in fresh nutrient medium via the bottom port (154)while the slower-settling detached cells and fast-settling, freshlydenuded microcarrier beads are held in circulation inside the settler,(ii) bare microcarrier beads (100-500 microns) will settle much fasterthan the stem cells (10-20 microns) and can be removed from the bottomport (154) while the stem cells are circulated in suspension, and (iii)finally the expanded stem cells can be harvested via the bottom port(154) at the desired concentration for subsequent cell therapyapplications.

Example 14: Co-Culture of Stromal Cells on Microcarrier Beads to Secretethe Necessary Growth Factors to Support the In Vitro Expansion or Growthof Other Differentiated Cells, Such as T-Lymphocytes or Cardiomyocytes

Growth and differentiation of pluripotent stem cells into cardiomyocytesor activated lymphocytes (CAR-T cells) require expensive growth factorsto be supplemented to the growth bioreactor. This cost can be reduced byco-culturing the desired cells with engineered mesenchymal stem cells(MSCs) that secrete the desired growth factors into the growth medium.These growth factor secreting cells support the growth of other desiredcells, such as CAR-T cells, cardiomyoctyes, etc. This co-culture can beeffected inside the bioreactor/cell sorter combination devices of thisdisclosure, and the cost of production or expansion of such cells issignificantly reduced. The expanded cells can be easily removed from theco-culture by feeding in fresh medium at a required flow rate to removethe expanded single cells or cell aggregates, while keeping larger,microcarrier beads inside the bioreactor/cell settler.

Example 15: Fractionation or Sorting of any Mixed-Cell Population, Suchas from Bone Marrow, into Several Distinct Sub-Populations withDesirable or Undesirable Characteristics

After loading the bioreactor/cell sorter device of the presentdisclosure with some initial bolus of a mixed cell population (such asbone marrow cells), we can feed in fresh nutrient medium at slow,step-wise increasing flow rates, such that the smallest cells (e.g.platelets, red blood cells, etc.) leave via top effluent stream at thelowest flow rates, followed by bigger cell types (lymphocytes,mononuclear cells, etc.) at increasingly higher flow rates, and then bythe biggest cell types (such as macrophages, megakaryocytes, etc.) atthe highest flow rates. By increasing the nutrient feed and the topeffluent flow rates at slowly-increasing step-wise flow rates,relatively pure populations of a single desired cell type is obtainedleaving the bioreactor/cell sorter device in a healthy cell culturegrowth medium so they can be propagated further for subsequent use.

Example 16: In Vitro Production of Universal Red Blood Cells

Novel genetic engineering methods are under development for directeddifferentiation of hematopoietic stem cells into erythroid cell lineage.Proerythroblast cells, the earliest committed stage in erhthropoiesis,are rather large (12-20 microns), up to three times larger than a normalerythrocyte. Polychromatophilic normoblasts, the subsequent stage inerythroid lineage, is smaller (12-15 microns) than the proerythroblastcells. Orthochromatophilic normoblast cells, the nucleated erythroidprecursor cells, are still smaller (8-12 microns), followed by the stillsmaller mature enucleated red blood cells. (Geiler, C., Andrade, I.,Clayton, A., and Greenwald. D. 2016, Genetically engineered in vitroerythropoiesis, International Journal of Stem Cells, 9: 53-59). Based onsize fractionation capabilities of the bioreactor/cell sorter devices ofthis disclosure, all the larger precursor cells are retained, and onlythe smallest mature enucleated red blood cells are removed from the topeffluent of the device, while all the larger precursor cells arecontinually expanding inside the bioreactor/cell sorter device.

Example 17: Large-Scale Platelet Production

Ex vivo expansion of high-ploidy megakaryocytic cells in controlledbioreactor culture conditions and their shearing off into smallerplatelet cells is increasingly understood at a fundamental level(Panuganti, S., Schlinker, A. C., Lindholm, P. F., Papoutsakis, E. T.,and Miller, W. M. 2013, Three-stage ex vivo expansion of high-ploidymegakaryocytic ells: Toward large-scale platelet production, TissueEngineering Part A, 19: 998-1014). As this understanding developsfurther, these necessary culture parameters can be obtained andcontrolled inside these bioreactor/cell sorter devices for growth anddifferentiation of megakaryocytic cells, while harvesting only themature, sheared off smaller platelets via the top outlet from thesettler.

To provide additional background, context, and to further satisfy thewritten description requirements of 35 U.S.C. § 112, the followingreferences are incorporated by reference herein in their entireties:U.S. Pat. No. 5,624,580, U.S. Patent App. Pub. 2009/159523, U.S. PatentApp. Pub. 2011/097800, U.S. Patent App. Pub. 2012/180662, U.S. PatentApp. Pub. 2014/011270.

The foregoing examples of the present disclosure have been presented forpurposes of illustration and description. These examples are notintended to limit the disclosure to the form disclosed herein.Consequently, variations and modifications commensurate with theteachings of the description of the disclosure, and the skill orknowledge of the relevant art, are within the scope of the presentdisclosure. The specific embodiments described in the examples providedherein are intended to further explain the best mode known forpracticing the disclosure and to enable others skilled in the art toutilize the disclosure in such, or other, embodiments and with variousmodifications required by the particular applications or uses of thepresent disclosure. It is intended that the appended claims be construedto include alternative embodiments to the extent permitted by the priorart.

What is claimed is:
 1. A settling device, comprising: a housingincluding: a first portion; a second portion; a cylindrical portionlocated between the first and second portions; at least one inlet forintroducing liquid into the housing; a first outlet port in the firstportion for harvesting a clarified liquid; a second outlet port in thesecond portion for harvesting a concentrated liquid; a sensor to measurea condition within the housing; and a stack of cones located within thehousing and occupying at least part of the cylindrical portion, eachcone of the stack of cones including a truncated apex and an open base,the truncated apex of each cone being oriented towards one of the firstoutlet port and the second outlet port, wherein the truncated apex ofeach cone defines a substantially central opening.
 2. The settlingdevice of claim 1, wherein the sensor is operable to measure at leastone of pH, dissolved oxygen (DO), and temperature within the housing. 3.The settling device of claim 1, wherein the housing is formed of aplastic, and wherein at least one cone in the stack of cones consistsentirely of a plastic.
 4. The settling device of claim 1, wherein anangle of inclination for a surface of a cone in the stack of cones isbetween about 30 degrees to about 60 degrees from vertical.
 5. Thesettling device of claim 1, wherein the at least one inlet is positionedbelow the stack of cones in the housing.
 6. The settling device of claim1, wherein the at least one inlet is associated with the second portionof the housing.
 7. The settling device of claim 1, wherein the secondoutlet port is formed at an apex of a conical end of the second portionat a lowermost portion of the housing.
 8. The settling device of claim1, wherein each cone of the stack of cones is spaced from an interiorsurface of the housing.
 9. The settling device of claim 1, wherein thesensor is positioned in the second portion of the housing.
 10. Thesettling device of claim 1, wherein the sensor comprises a fluorescentprobe.
 11. A method of settling particles in a suspension, comprising:(a) introducing a liquid suspension of particles into a settling devicewhich includes a housing having: a first portion; a second portion; acylindrical portion located between the first and second portions; atleast one inlet for the liquid suspension to enter the housing; a firstport for harvesting a clarified liquid; a second port for discharging aconcentrated liquid suspension; a sensor to measure a condition withinthe housing; and a stack of cones located within the housing andoccupying at least part of the cylindrical portion, each cone of thestack of cones including (i) a truncated apex, and (ii) an open base,wherein the truncated apex of each cone is oriented toward one of thefirst portion and the second portion, wherein the truncated apex of eachcone defines a substantially central opening; (b) collecting theclarified liquid from the first port; and, (c) collecting theconcentrated liquid suspension from the second port.
 12. The method ofclaim 11, wherein the liquid suspension comprises at least one of arecombinant cell suspension, an alcoholic fermentation, a suspension ofsolid catalyst particles, a municipal waste water, industrial wastewater, bacterial cells, yeast cells, plant cells, algae cells, plantcells, mammalian cells, murine hybridoma cells, stem cells, CAR-T cells,red blood precursor and mature cells, cardiomyocytes, yeast in beer, andeukaryotic cells.
 13. The method of claim 11, wherein the liquidsuspension comprises recombinant microbial cells selected from at leastone of Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces lactis,Aspergillus niger, Escherichia coli, and Bacillus subtilis, and whereinthe clarified liquid includes secreted proteins or enzymes.
 14. Themethod of claim 11, wherein the liquid suspension comprises one or moreof microcarrier beads, affinity ligands, and surface activatedmicrospherical beads.
 15. The method of claim 11, wherein introducing aliquid suspension comprises directing the liquid suspension from aplastic disposable bioreactor bag into the settling device.
 16. Themethod of claim 11, wherein the clarified liquid collected comprises atleast one of: biological molecules, organic or inorganic compounds,chemical reactants, chemical reaction products, hydrocarbons,polypeptides, proteins, alcohols, fatty acids, hormones, carbohydrates,antibodies, glycoproteins, terpenes, isoprenoids, polyprenoids, beer,biodiesel, insulin, brazzein, antibodies, growth factors, colonystimulating factors, and erythropoietin (EPO).
 17. The method of claim11, further comprising: removing dead cells from the housing through thefirst port, wherein the first port is formed through the first portion;and introducing a nutrient medium into the housing through the secondport.
 18. The method of claim 11, wherein the sensor comprises afluorescent probe, and wherein the sensor is further operable to measureat least one of pH, dissolved oxygen (DO), and temperature.
 19. Themethod of claim 11, further comprising introducing at least one of O₂,CO₂, and N₂ into the housing to alter one or more of a pH level and aconcentration of dissolved oxygen within the housing.
 20. The method ofclaim 11, further comprising using data received from the sensor toadjust one or more of pH, dissolved oxygen concentration, and pCO₂within the housing.